GENE/BICH 432: MOLECULAR
GENETICS LABORATORY
Course Schedule and Syllabus for Spring 2004
Jan 21 Introduction to class and
Module 1-Cloning and Expression of A Known Gene Product
Jan 26 Restriction digestion of DNA
Jan 28 Gel analysis of DNA - SHOW and TELL – Bring a scientific journal article that has cloning and immunoblot results
Feb 2 Gel purification of DNA and ligation
Feb 4 Transformation of bacteria and analysis of ligation products - Practice Methods Writing
Feb 9 Bacterial cultures –Edit Methods
Feb 11 Minipreps of plasmid DNA by alkaline lysis - Writing Assignment #1 –Methods
Feb 16 Restriction analysis of plasmids and Quantitation of plasmid DNA –
Feb 18 Transform a pUB-GX2 clone into JM109(DE3) - Practice Results Writing
Feb 23 Grow recombinant cells expressing UB protein
Feb 25 Inducing the production of UB protein and making and starting SDS-PAGE - Edit Methods and Results
Mar 1 Make the SDS-PAGE gel and UB protein samples –
Mar 3 Run the SDS-PAGE and Western blotting
Mar 8 Block and incubate the blot with primary antiserum –
Mar 10 Immunodetection of UB protein and Discussion of results for Module 1 - Writing assignment #2 – (Methods and) Results
Mar 15 and 17 – Happy SPRING BREAK!!!
Mar 22 Introduction to Module 2 – De novo cloning and Identification of a Gene Product - REVISED!!! (Module 1 report (abstract) due)
Mar 22 to May 3 (M and W from
Ap 14 – Writing Assignment #3 due – Methods and Results for Module #2
May 5 Discussion of Module 2 and Summary
May 7 Writing Assignment #4 due – Proposal for engineered expression in a mammalian cell. Turn in lab notebooks also (Kleberg Rm. 410).
Students get individual experience with non-radioactive detection of specific proteins and working with DNA modifying and synthetic enzymes. We will work with recombinant gene expression systems in E.coli and in mammalian cells that are driven by plasmid cloning vectors. Analytical techniques include electrophoresis on agarose (for DNA) and acrylamide gels (for protein) and utilize PCR for cloning and sequencing.
Instructor: Dr. Nancy Ing
Office: Kleberg 410D
845-3560
ning@cvm.tamu.edu
Office Hours: TR
Teaching Assistants:
Kelly Vaughan
lkvaughan@tamu.edu
and
Yulia Surovtseva
email syv@neo.tamu.edu
Description: Laboratory for molecular genetics providing technical experience with tools of molecular biology
Prerequisistes: BICH 431 or concurrent
registration
Grading:
10 % for each Writing assignment #1 to #4
This class is also being developed as a “W” or Writing course so will involve formal (writing assignments) and informal writing (lab notebooks) in scientific style. The writing assignments will be graded on a scale:
10 pts – Good presentation of scientific information in a concise form that is consistent with standards of writing discussed.
8 pts – Fails to meet the highest criteria in information or form.
6pts – Fails in information, clarity and style and overall communication.
30% Notebook
Lab notebooks are bound volumes, are kept in pen with numbered, dated pages. They are designated only to that purpose, are labelled on the cover with Name, dates and lab, and are only removed from the lab with instructor permission. In them, each person describes their activities and observations each day. Each page should be dated. Each photo or X-ray film should be labeled with initials, date, and identification. Record things chronologically along with dates. Start a new project on a new page with a description of its purpose. A stranger should be able to pick up your notebook and understand how and why you did an experiment. Protocols don't have to be written each time, but may be referred to, instead. For this, you may consider the handouts you receive in lab as references. Use them as such and refer to specific pages (dates) within them. Record your activities and observations clearly, using complete and understandable sentences. Write down your reagents, including buffers and buffer recipes. KEEP UP WITH NOTEBOOK ENTRIES EVERY DAY…otherwise, data will be lost!
Daily notebook entries (including answers to questions posed in these protocols) will be graded at least four times in unannounced spot checks and at the end of the class and they are turned in with the Module 2 report. They are graded on information (8 points) as well as quality of writing (2 points) for example, complete sentences, appropriate word usage, etc.). Each check is worth 10 points and the highest 3 will be used for the notebook part of the final grade.
30% Participation (see LAB RULES), Preparation (COME PREPARED!) and Results
Attendance is mandatory (No makeup labs)
Loss of one letter grade in the participation grade for each unexcused missed lab.
Lab notebooks that are missing are
easy to grade! 0!
Loss of 10 points on written assignments for each
calendar day past due (
Activities:
Classes meet MW 1 to 3:40 in Biochemistry 243. The goal of the class is understanding the experiments performed and their purposes and ultimately being able to use them. Classes include discussions and videotape presentations of theories and practical aspects. Scientific writing will be used to record and report activities and results.
Suggested Reading
TEXT: Molecular Biology made simple and fun, D.P.Clark, L.D.Russell, Cache River press 1997
ISBN 0-9627422-9-5
You may choose to read the book from front to back. I recommend especially the first nine chapters. Then you can review the chapters below before lab.
Jan 24 Ch. 9 Messing about with DNA
Feb 6 Ch. 8 Sex among the low-lifes and its exploitation
Feb 13 Ch. 2 Bacteria: the Molecular biologist’s Guinea Pigs
Feb 16 Ch. 19 Gene creatures: Viruses and plasmids
Feb 23
Feb 27 Ch. 7 Protein: the buck stops here
Mar 20 Ch. 16 Just do it! The techniques of Molecular biology
Mar 19 Ch. 4 Required reading: the molecular basis of heredity
Ap 8 Ch. 17 PCR the polymerase chain reaction
Ap 16
BICH/GENE 432
Safety Rules
1. ABSOLUTELY no food or drink (or containers) in the lab. This includes gum.
2. Always wear gloves when working with Ethidium bromide, acrylamide and organic chemical compounds, as well as biohazard (E.coli) materials.
3. Always wear UV safety glasses when using UV illumination, or work behind UV shields provided.
4. Use special care when working with open flames. Don't forget to turn off gas after use. NO UNATTENDED FLAMES!!!
5. Clean up spills immediately. Notify instructor if hazardous compounds are spilled.
6. Discard organic chemical solutions in appropriate bottles.
7. Discard ethidium bromide waste in correct container.
8. All culture medium and labware used for bacteria (“biohazard”) needs to be autoclaved or put in Chlorox before disposal or washing.
9. After using radioactive materials, wash the work area and clean spills immediately. Dispose of gloves in radioactive waste as soon as you are finished.
10. All sharps (including broken glass, needles and razor blades) should be disposed in clearly marked containers, not in the general trash.
11. If you have questions about anything, ASK!
12. Wearing lab coats and safety glasses (if other glasses are not worn) is required!
13. Shoes must be closed-toe (no open-toed sandals).
LAB RULES
WELCOME TO THE LAB!
Follow these guidelines so productivity and fun can be maximized.
A. GENERAL
1. Everyone is individually responsible for the experiments. Come prepared by reading protocols and required reading in advance!! Activities will be started immediately, while explanations and discussion sessions will occur as time permits. COME PREPARED to ASK QUESTIONS, especially during discussions.
2. Equipment in this and neighboring labs is shared. Know or ask how to use it. Obey user rules, such as signing logs. Leave all equipment in good working order. If there are problems, tell someone so we can fix them!
3. Leave the lab better than you found it. Wash your own glassware, clean up your work area, write the names of reagents that are running out on the "to be ordered" list, etc.
4. Lab notebooks are bound volumes, are kept in pen with numbered, dated pages. They are designated only to that purpose, are labelled on the cover with Name, dates and lab, and are only removed from the lab with instructor permission. In them, each person describes their activities and observations each day. Each page should be dated. Each photo or X-ray film should be labeled with initials, date, and identification. Record things chronologically along with dates. Start a new project on a new page with a description of its purpose. A stranger should be able to pick up your notebook and understand how and why you did an experiment. Protocols don't have to be written each time, but may be referred to, instead. For this, you may consider the handouts you receive in lab as references. Use them as such and refer to specific pages (dates) within them. Record your activities and observations clearly, using complete and understandable sentences. Write down your reagents, including buffers and buffer recipes. KEEP UP WITH NOTEBOOK ENTRIES EVERY DAY…otherwise, data will be lost!
5. All reagents and samples should be saved and must be labeled with the date; your initials, and WHAT IT IS. In my lab, we use simple sample numbers, such as 1 - 10. To key it into our notebook, we write: lab notebook #, page #; (NI2P30 relates to Nancy Ing Book 2, page 30), so the notebook, not the tube, is where the full description of the sample exists. Items not labeled sufficiently may be discarded.
6. Store things in appropriate places! For plasmids and reactions and buffers, store at –20C in storage box provided to your group unless otherwise noted. Note storage places in your notebook.
B. SAFETY IS THE #1 PRIORITY
1. The only safety activity not strictly enforced is wearing safety glasses: this is a good idea but is not mandatory. Wearing a lab coat is mandatory and wearing gloves will become a habit (see below).
2. Working with open flames and hazardous chemicals have strict safety protocols - ask for them and follow them.
3. Working with radioactivity is a privilege, not a right. Workers must monitor for contamination, before, during, and after the procedure. Radiation safety training is required. WE RUN A CLEAN LAB.
4. Because we work with HAZARDOUS SUBSTANCES, there is NO EATING, DRINKING, SMOKING, or APPLYING MAKE-UP in the lab.
5. Garbage must be disposed of properly. Glass and sharps, biohazard, chemical, and radioactive waste must be separated from the rest.
C. GOOD LAB TECHNIQUES
1. ICE IS NICE! Work on it unless otherwise directed. It slows degradation of macromolecules.
2. Many reagents settle on storage, so mix them! All frozen solutions need to be thawed and mixed before using.
3. ENZYMES DO OUR WORK. They are stable as glycerol solutions at -20oC. Keep them in the freezer as much as possible. Only remove them in -20oC blocks. DO NOT WARM ENZYME STOCKS! When pipetting small amounts of viscous solutions like enzymes, check loaded pipet tip and evacuated one to assure that enzyme got into the reaction. After addition, mix reaction solution gently but thoroughly: can pipet total volume up and down OR vortex gently and flash spin to return reaction to the tube bottom.
ADA Statement, Copyrights, and Plagiarism
The Americans with Disabilities Act (ADA) is a federal antidiscrimination statute that provides comprehensive civil rights protection for persons with disabilities. Among other things this legislation requires that all students with disabilities be guaranteed a learning environment that provides for reasonable accommodation of their disabilities. If you believe you have a disability requiring an accommodation, please contact the Dept. of Student Life, Services for Students with Disabilities in Room 126 of the Koldus Bldg. or call 845-1637.
Copyrights
The handouts used in this course are copyrighted. By
handouts”, I mean all materials generated for this class, which include but are
not limited to syllabi, quizzes, exams, lab problems, in-class materials,
review sheets, and additional problem sets. Because these materials are
copyrighted, you do not have the right to copy the handouts unless I expressly
grant permission.
Plagiarism
As commonly defined, plagiarism consists of passing off as one’s
own ideas, words, writings, etc., which belong to another. In accordance with
this definition, you are committing plagiarism if you copy the work of another
person and turn it in as you own, even if you should have the permission of
that person. Plagiarism is one of the worst academic sins, for the plagiarist
destroys the trust among colleagues, without which research cannot safely be
communicated.
If you have any questions regarding plagiarism, please consult the latest issue of the Texas A&M University Student Rules, under the section “Scholastic Dishonesty.”
WRITING
A PAPER FOR A SCIENTIFIC JOURNAL
by Linda Guarino
TITLE - This is the most important part of a manuscript. A reader begins here, and will also finish here if the title does not promise a subject of interest to him/her. A good overall rule is to use the fewest possible words that adequately describe the contents of the paper. But, do not sacrifice words for specific information. For example 'DNA cloning' is a short title, but it is too general. A popular trend in recent years is to publish papers where the title is a complete sentence that summarizes the major conclusion of the manuscript. Personally, I prefer titles that describe the work, not the results.
ABSTRACT - An abstract is a mini version of the paper. It should provide a brief (less than 250 words) summary of the major points of the manuscript. The abstract should state the objectives, describe the methodology used, summarize the results, and state the principle conclusions. The abstract should be written in the past tense, because it refers to work done.
INTRODUCTION - First of all, state the nature and scope of the problem investigated. Review the pertinent literature (THIS IS NOT REQUIRED IN THE GENE/BICH 432 CLASS). Describe the method of the investigation. State the principle results. State the principle conclusions suggested by the results. The first two parts should be in present tense, while comments relating to the present study should be in past tense.
METHODS - The methods section should expand upon the description of
the methodology that was presented in the abstract. The order of presentation
is usually chronological (methods used in initial stages of the study are
presented first). However, sometimes it makes more sense to group similar
methods into sections, even though they were not used at the same time. Due to
space limitations in journals, methods are not usually described in detail if
they have previously been published. If a scientist uses a protocol that is
identical to one previously described, he/she would state 'The DNA was prepared
according the procedure previously described (reference). If there were minor
differences, he/she would state 'according to the procedure of (ref.) with
minor modifications' and then describe the modifications. In this class, you
may assume that the class protocol has been published. Therefore you don't need
to give the details, but you need to describe the general strategy. For
example, you should say 'the DNA was purified by the alkaline lysis procedure as previously described' not 'the DNA was
purified as previously described'. In addition to the class protocol, you could
also reference the Molecular Cloning manual or the Promega
manual. The methods section should be written in past tense.
RESULTS - The results section is a text presentation of the data. It should lead the reader through the figures, pointing out important information. All important data should be given in figure form. Figures should have complete legends - so that they can be understood without reading the paper. The figure legends may have redundancy with results text.
DISCUSSION - The discussion should put the results into perspective. Discuss the results without recapitulating the results section. Show how your results and interpretations agree. State your conclusions clearly, and summarize the evidence for each conclusion. Selection of correct tense is more difficult in the discussion than in the other sections. Your own work should be described in past tense. If reference is made to published work, it should be in present tense.
Suggested reading:
Day, R. A. 1988 How to write and publish a scientific
paper, 3rd ed. Oryx Press,
GENE/BICH 432 ning
MOLECULAR GENETICS LABORATORY MANUAL
Jan 21 Introduction to class, lab safety and micropipettors
Module 1-Cloning and Expression of A Known Gene Product
In this module, we will subclone a cDNA encoding bovine ubiquitin (bUB) protein from a general use plasmid vector into a plasmid vector designed for expression (transcription and translation) of the cDNA in vitro. The cDNA insert will be excised from pbUB by restriction digesting with Sac I (cuts at GAGCT/C) and Xho I (cuts at C/TCGAG, see Fig. 1). Simultaneously, the vector pGEMEX-2 will be digested with the same enzymes. In this way, sticky EcoR I and Xho I ends will be generated on insert and vector, so that they can be ligated. This will allow directional cloning of the insert into the vector. Agarose gels (non-denaturing) will be used to analyze DNA size, concentration, and shape, as well as to purify DNA.
After ligation, the new plasmid DNA is introduced into E. coli cells to be amplified there. Plasmid "minipreps" will be purified from liquid cultures of single bacterial colonies. Restriction digestion will identify clones that carry the desired plasmid. The plasmid will be transformed into E coli cells that will overproduce the protein by transcribing and translating the cDNA. The E.coli cells will be broken and their proteins will be analyzed on denaturing polyacrylamide gels: (SDS-PAGE). Proteins will be blotted to filter membranes ("Western blots") for analyzing with antibodies to the ubiquitin protein. A chemiluminescent development system will allow visualization of bands as ubiquitin protein.
Pipetting Exercise:
Your success in molecular genetic experiments is dependent upon your ability to pipet accurately and repeatably. They are expensive and breakable! Make the micropipettors your friends and test their range of uses in the exercises below:
1. Read the instructions in the Appendix
2. Weigh 1000 ul of water in a 1.5 ml tube. Repeat three times. Weigh on a scale with tared to a 1.5 ml tube. What is the density of water? _______ g/ml
3. Repeat above with isopropanol. What is the density of isopropanol? _________g/ml
4. Measure the volume of the unknown sample using your appropriate pipettor.
(Set it to 0, submerge the disposable tip, and dial up until the solution completely
enters the tip. Read the volume from the dial.)
My unknowns, labeled: A was _____ul.
B was ____ul.
C was ____ ul.
The data from #2 to 4 should be recorded in your notebook.
DISCUSS: DNA Structure using human nucleotide models
- Make a six base DNA strand (random sequence)…Identify 5’ and 3’ ends. What type of bonds exist between bases?
- What is the chance that a specific six base sequence will occur?
- Make a complementary DNA strand hybridized to the one in #1. How are the strands oriented to each other? What type of chemical bonds exist between the DNA strands?
- Reverse the 5’ to 3’ direction of the complementary strand. What bonds (if any) did you have to break to do this? How is this strand related to the initial strand?
- Make an EcoR I restriction enzyme site. What bonds does the enzyme cut? What makes the ends "sticky"? Do the ends have 5’ or 3’ overhangs or one of each?
WATCH VIDEO: RESTRICTION ENZYME DIGESTION
Jan 26 Restriction digestion of DNA (vector and insert)
A. (Each student do all reactions) To prepare bUB insert (530bp) and pGEMEX-2 vector (4000bp) DNAs from plasmids for subcloning, we'll restrict each with Sac I and Xho I. We’ll also do controls for pGEMEX-2 restriction with Sac I alone and Xho I alone. We’ll do reactions of 40 ul, to generate duplicate 20 ul samples, a volume easily loaded on a gel.
As for all enzyme reactions, BE SURE YOUR CALCULATIONS AND ADDITIONS TO EACH TUBE ENSURES THAT THE ENZYME WILL BE FUNCTIONAL. Write out your reaction components in the order to be pipeted, check them with an instructor, and pipet accurately. Keep track of additions by checking them off as you add them or by moving the reagent tubes.
1. Calculate 5 µg of pbUB insert plasmid and 0.5 ug pGEMEX-2 vector plasmid. Pipet volumes into 1.5 ml tubes: one for pbUB and three for pGEMEX-2.
2. Calculate amount of H2O to make the final reaction volumes each 40 ul (a double-size reaction). Since digestion requires 5 to 10 units of enzyme per ug DNA, the pbUB tube will need 25 to 50 units while the pGEMEX-2 tubes need only 5 to 10 units of each enzyme: both or one enzyme in each tube. Since most enzymes are in glycerol stocks at 12 units/ul, estimate 3 ul and 1 ul for pbUB and pGEMEX-2 reactions, respectively.
But, maximum enzyme volume in any reaction is 10%!!!!!!!!! (because they are in 50% glycerol stocks, which inhibits the reaction at higher levels), so we only get to add 2 ul each.
10X Buffer is 4 ul in each (see 3). Add appropriate volume of water to
tubes.
|
|
|||||
|
tube # |
DNA(ul) |
nanopure water(ul) |
10x buffer |
enzymes |
Total (ul) |
|
1 |
___ul pbUB |
____ul H2O |
4 ul, ? |
2 ul Sac I, 2 ul Xho I |
40 ul |
|
2 |
___ul pGEMEX-2 |
____ul H2O |
4 ul, ? |
1 ul Sac I, 1 ul Xho I |
40 ul |
|
3 |
___ul pGEMEX-2 |
____ul H2O |
4 ul, ? |
1 ul Sac I |
40 ul |
|
4 |
___ul pGEMEX-2 |
____ul H2O |
4 ul, ? |
1 ul Xho I |
40 ul |
(The last two are controls for #2. Why don’t we need a similar controls for reaction #1?)
3. Add 4 µl of the appropriate 10X buffer. It should be one in which both enzymes can have 100% activity. Choose this from the tables in the spiral bound Promega Protocols book. All working concentrations of buffer are "1 X".
4. Add 10 units per ug DNA of the restriction enzymes Sac I and Xho I (see calculations in #2, above).
NOTES ON USING ENZYMES: These are the expensive workhorses of molecular biology. Your success depends on their function. Therefore, they should be handled with care: KEEP THEM AT -20oC at all times by setting up the entire reaction prior to retrieving them from the freezer. Keep them in the -20oC blocks while pipeting. They are in glycerol, so are viscous. Visually check that enzyme is picked up in the pipet tip and delivered into each tube. Ensure their purity by using new pipette tips each time!!!! After addition, mix the enzyme into the reaction by gently pipeting the whole reaction up and down, or by vortexing and flash microfuging.
5. Incubate for 1 h at the appropriate temperature (37oC). During this time, make gel (below) and make one uncut plasmid sample of 0.5 ug: (X ul DNA plus TE buffer to 10 µl volume, then add 1 µl 10X DNA dye.) Store the remainder of the plasmids and the uncut sample at -20oC.
6. At the end of the incubation, split the reaction into two tubes. Add 2 ul 10X DNA dye to each. Store at -20oC.
Make an Agarose Gel electrophoresis (2 students per gel).
1. Make 1 liter of 1X TAE for each gel
2. Make 100 mls of 1% agarose gel + 0.5 µg/ml EtBr in 1X TAE: Combine 100 mls 1X TAE, 1 g agarose, and ___ ul of 10 mg/ml EtBr in a 250 ml beaker. Cover with plastic wrap. Bring to boiling 2 to 3 times in a microwave oven. Agarose should be IN SOLUTION!!!! (solution perfectly clear…no clumps!
3. After cooling to 60° C, pour a mid-size gel and use two 14-well combs. Follow the set-up instructions of the TA for your specific gel unit!!!
4. Store the gel at 4° C under a small amount of buffer in a wrapped container.
WATCH VIDEO: AGAROSE GEL ELECTROPHORESIS
DISCUSS
1. What is a restriction enzyme? What is a unit?
2. What are the components in optimized buffer?
3. Why do we use water baths and not air incubators?
4. Why are we doing controls 3 and 4?
NOTES: Check bath temperatures always before and during use!!!
In NOTEBOOKS define reagents such as TAE. Find references in your text and Promega Protocols books and note their locations in your notebook.
Jan 28 Agarose Gel Electrophoresis
IF you have not loaded an agarose gel before, practice loading 1X DNA dye in the wells before you load samples. When you are comfortable with the procedure, you can flush the dye out with a transfer pipet and use the wells for samples.
1. Load gel with uncut plasmid (1 sample) and 4 plasmid digests (20 ul each… save the duplicate reactions!). Include Lambda Hind III EcoR1 markers (1 µg) in one well of top and bottom rows of wells or at each gel loading if loadings occur at different times.
2. Run at 120V, 1 h.
3. Photograph on UV light box.
4. By comparing the bands between lanes (controls and experimental), one can describe (see last page of Appendix)
a. The molecular size of the fragment
b. The [DNA] of each fragment, and
c. the molecular form (circular vs. linear) of the fragments
For (a), compare migration distance to that of Lambda DNA standard fragments - estimate bp size
For (b), compare brightness of bands to those of Lambda fragments - estimate ng DNA. Divide by volume of the sample loaded to get [DNA] in the original sample. NOTE: This is often more reliable quantitation than A260 measures!!!
For (c), note that uncut plasmids run as two forms, fast supercoiled and slower nicked circular. The restricted plasmid from vector only transformation shows that linear DNA is slower or intermediate in migration speed. Since markers are linear DNAs, their migration only relates to other linear DNAs. So supercoiled and nicked circular forms do nto migrate true to their size. However, they may be components of restriction digests if digestion is incomplete.
SO COMPARE ONLY LINEAR DNA TO LINEAR DNA MARKERS!!!
DISCUSS...1. How can you tell if a restriction digest is complete?
2. What are the expected results?
3. Why a 1% gel? Why not a 0.4 or 4% gel?
WRITE IN YOUR NOTEBOOKS:
Describe your experimental (gel) results completely (pretend there is no gel picture…there was science before Polaroid film!) Be complete.
Write conclusioins/plans: Which pUB and pGEMEX-2 restrictions are you going to use next time? Why?
WATCH VIDEO: DNA LIGATION REACTION
Feb 2 Gel purification of DNA on low melt gels and ligation
(During this lab, TAs will make LB and 500 LB agar: Autoclave to sterilize and melt the agar. Cool the LB agar to 60 ° C. Add ampicillin to 100 ug/ml and pour into 100 mm plates. After solidifying, invert them. Leave them on bench to dry.)
DNA Ligations are the weak point in most cloning procedures. Here we are set up for success by using "sticky ends": restriction sites with 4 base overhangs that efficiently bind to each other (EcoR I to EcoR I and Xho I to Xho I). We'll purify the fragments on low melt agarose gels and do the ligation directly in excised chunks of low melting agarose to minimize loss of DNA.
1. For each four students, make a 0.8% low melt agarose gel + EtBr (as above).
NOTES: These are like soft jello - Hard to handle!! Put tabs of tape under comb suports to raise the well bottoms further from the bottom surface of the gel mold. Cool completely and Be Careful removing combs!!! Test representative or all wells for breaks at the bottom by loading DNA dye. IF OK, flush wells and reload with sample.
2. Each group of 2 students should use the best DNAs in Tubes 1 and 2 from the previous lab: highest concentrations in desired bands and choose tube 2 from the best cut vector controls (tubes 3 and 4). Make sure you’ve added 2 µl 10X Loading dye. Run at 100 V for 30 min.
3. Cut out bands at expected sizes (530 bp for pbUB insert and 4000 bp for pGEMEX-2) and remove excess agarose on the bottom and sides of gel chunk while visualizing under Longwave (low energy) UV light. This minimizes damage to the DNA that would interfere with amplification in E.coli. Put gel chunks in individual, clean, labeled 1.5 ml tubes.
4. Melt DNA + agarose 70oC, 10 min. Then keep it at 37 oC for 5 min or until samples are pipeted out.
5. In ligations, the larger concentration of insert DNA is used to drive the success of the reaction. Each group of 2 students will make 3 ligation reactions: All three have 1 ul vector: one with maximal levels of insert DNA (8 ul, "MAX"), one with minimal levels (3 ul, "MIN"), and one lacking insert DNA ("negative control").
Combine H2O and DNA (in agarose or in solution) to a 9 µl volume in the 37oC block:
|
|
|||
|
tube |
Vector DNA |
Insert DNA |
H2O |
|
MAX |
1 ul pGEMEX vector |
8 ul pbUB insert |
none |
|
MIN |
1 ul pGEMEX vector |
3 ul pbUB insert |
5 ul |
|
Negative Control |
1 ul pGEMEX vector |
none |
8 ul |
6. Prepare a "master mix" of ligase and buffer on ice. Have three reactions so make for four reactions to ensure enough:
2 µl 10X Ligase Buffer X 4 = 8 ul
1 µl T4 DNA Ligase X 4 = 4 ul
8 µl H2O X 4 = 32 ul
Combine and mix components gently but well.
"MASTER MIXES" ARE USED FOR SIMILAR SETS OF REACTIONS. They optimize consistency between reactions as well as save time and can increase accuracy in pipetting.
7. Add 11 µl ice-cold ligase mix to each reaction tube. Finger flick the tube immediately and slam it on ice. Reactions will gel while ligation occurs. Incubate 15oC, overnight (O/N).
DISCUSS: Theory of low melt gel reactions.
- Why the control?
- What are expected results?
Feb 4 Transformation of bacteria and analysis of ligation products
Notes:
- USE STERILE TIPS, etc.
2. DISCARD WASTE CONTAMINATED WITH LIVE BACTERIA IN BIOHAZARD BAGS!!!!!!!!!!!!!
A. Transformation of Bacteria
"Bacterial transformation" relates to the change of the bacterial phenotype by introducing a plasmid containing an antibiotic resistance gene. "Competent cells" are made receptive to plasmids by making their membranes permeable with by calcium treatment (abuse). Plasmids adhere to the fragile cells, enter on heat shock, and resistant cells selectively grow on plates containing the antibiotic after a 1 h recovery period in broth culture lacking antibiotic.
Most plasmids used for cloning purposes encode an antibiotic resistance gene. This trait is useful for selection of bacterial cells harboring plasmids against a large background of cells that do not. When cells are plated in the presence of the appropriate antibiotic, only those cells containing plasmids can grow. After initial selection, it is necessary to maintain cells in the presence of the selective agent to prevent loss of the plasmid. In both pGEMEX and pBluescript vectors, the selection system is based on the ß-lactamase gene which encodes an enzyme for resistance to penicillin family of drugs, like ampicillin.
All plasmids contain sequences for origin of DNA replication. This sequence is essential to ensure that plasmids are replicated and passed to daughter cells. Many popular plasmids are based on the ColE1 origin. This is a high copy number plasmid because replication is not linked to chromosomal replication. Plasmid number is amplified at stationary phase resulting in 300 - 500 copies per cell. This is extremely useful in isolating large amounts of plasmid, even from small cultures of E. coli.
Most plasmids utilize a color screening system based on the ß-galactosidase gene. The plasmids contain a fragment of the LacZ gene. This encodes a peptide that is required for the formation of biologically active ß-gal tetramers in strains of E.coli that have a defective lacZ gene. This is called a-complementation. In the absence of plasmid, these strains cannot form active enzyme. The presence of plasmid can be detected by plating cells in the presence of the chromogenic substrate, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal). Active ß-gal is formed in cells with plasmids, and the enzyme hydrolyzes X-gal yielding a blue precipitate. This makes the colonies turn blue. The cloning sites are located within the LacZ gene on the plasmid. Therefore, plasmids containing inserts do not complement the host protein, and the colonies remain colorless or white. So in addition to growth selection on the plates (selects for E. coli containing plasmid), individual colonies can be selected for color: those containing an insert (white) are desired while those with recircularized vectors lacking inserts (blue) are not.
Each group of 2 students performs one transformation for each ligation (3 per group) and one positive control transformation with 10 ng of circular plasmid DNA.
1. Thaw 500 ul JM 109 competent cells on ice (20 min.).
2. Bacterial cells are heavy and settle as the tube rests. Gently resuspend the cells before pipeting. Pipet 100 µl into a four ice-cold 1.5 ml tubes, labeled according to ligations and one extra labeled "+" control.
3. Spin ligation tubes to collect contents at the bottom of the tube. Melt ligations at 70oC, 10 min. During this time, each group should choose a circular plasmid DNA from those given out and dilute a small aliquot of it to 10 ng/ul. The fourth transformation will use 1 ul of this as "+" control.
4. Cool ligations to 37oC in block, 5 min.
5. Add 1 µl ligation or "+" control plasmid DNA to cells; mix by pipeting up and down. (Save rest of ligation at 37 ° C for B, below)
6. Incubate on ice 30 min. Mix tube contents every 10 min. by tapping gently.
7. Heat shock 42oC, 45 sec. No shaking.
8. Cool transformations on ice for 2 min.
9. Add 400 µl of room temperature (RT) S.O.C. broth.
10. Agitate cultures gently, 37oC, 1 h. (watch video and discuss now! See below)
11. For the transformations from ligations, spread all 500 µl on a LB + Ampicillin plate. For the transformation from circular plasmid DNA, spread only 50 ul. LABEL CULTURE PLATES ON THE BOTTOM, because plate tops can get shuffled. Keep plates upside down to minimize water dripping off lids onto colonies.
12. Incubate inverted plates at 37oC O/N.
B. Analysis of ligation products
1. Make a 1% agarose gel - DO THIS EARLY IN CLASS
2. Add 10X DNA loading dye (___ul) to sample from A4.
3. Load samples and DNA marker. [ Optional: Can run along with uncut and cut plasmid controls.]
4. Run gel 45 min at 100 V.
5. Record results in text style. Photograph is optional.
WATCH VIDEOS : BACTERIAL TRANSFORMATION,
DISCUSS....
What do you expect to see on your gel? On your plates?
Tell me why we didn’t
include X-gal in the plate agar? (Why no blue-white
selection?)
* genotype of JM83 is :
Sup E thi D (lac-proAB) F’[traD36 proAB+ lacIq lacZ D M15]
Feb 9 Bacterial Cultures
Don’t forget to record your observations of your bacterial plates.
To identify clones for the desired plasmid (pbUB-GX2), individual (clonal) colonies will be amplified in liquid culture and plasmid will be prepared.
Each student group will grow 6 cultures from 6 colonies on either the "MAX" or "MIN" agar plate.
Use sterile technique !!!!
1. Pipet 5 mls LB broth into 6 sterile culture tubes.
2. Add ampicillin stock to make broth contain 50 ug/ml.
3. Flame a loop and allow it to cool in air (30 sec). Then cool it by touching the plate where no colonies are growing.
4. Touch an individual colony to pick up its cells.
5. Submerge the loop in the broth in a labeled tube. Repeat steps 3 to 5 for all tubes.
6. Incubate O/N at 37° C with vigorous shaking.
7. After incubation, cultures will be refrigerated until use.
8. Wrap Parafilm around agar plate edges and store plates upside down at 4° C.
Watch Plasmid DNA Miniprep
video.
Feb 11 Minipreps of plasmid DNA by alkaline lysis
DISCUSS....Results of analysis of ligation products gel and the expected results in colony numbers here.
What’s the efficiency of transformation?:
You put in 10 ng plasmid in "+" control transformation. You transform 1/20 of that and plate 1/10 of that. So plated cells were exposed to 0.05 ng of circular plasmid...
0.05 ng divided by 3500bp plasmid * 700g/bp = 2 * 10–17mol
and 2 * 10–8 mol * 6.022 * 10E23 molecules/mol (Avogadro’s number) = 1.2 * 107 transformants possible growing on the positive control plate.
WARNING! PHENOL IS CORROSIVE! DON’T GET IT ON YOUR SKIN!!!
In a short, mind-numbing period of tube shuffling, one can extract plasmid DNA for analysis of desirable clones
1. Spin down 1.5 ml overnight culture cells in a 1.5 ml tube - (DISCARD all materials from steps 1 -3 that are contaminated with cells in BIOHAZARD BAGS.)
2. Save the rest of the overnight culture at 4oC - IF it is a good clone, you'll want to make a glycerol stock for long term storage and a streak plate for short term use.
3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.
4. Resuspend the pellet by trituration (pipetting up and down) in 150 µl of an ice-cold solution ("I")of 50 mM glucose + 10 mM EDTA + 25 mM Tris · HCl (pH 8.0) + 4 mg/ml lysozyme (last added fresh)
5. Store for 5 minutes at room temperature. The top of the tube need not be closed during this period.
6. Add 300 µl of a room temperature solution ("II") of 0.2 N NaOH + 1% SDS.
Close the top of the tube and mix the contents by inverting the tube rapidly two or three times. Do not vortex. Store the tube on ice for 5 minutes.
7. Add 225 µl of an ice-cold solution ("III") of potassium acetate (~pH 4.8) made up as follows: To 60 ml of 5 M potassium acetate, add 11.5 ml of glacial acetic acid and 28.5 ml of H2O). The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate.
Close the cap of the tube and vortex hard, put on ice, vortex again. Store on ice for 5 min.
8. Centrifuge for 15 minutes in an micro-centrifuge at 20o C.
9. Transfer 600 µl supernatant to a fresh tube. (Avoid all white, solid precipitated genomic DNA and protein).
10. EXTRACT WITH Phenol/Chloroform. This means: Add an equal volume of phenol/chloroform pH8. Mix by vortexing. After centrifuging for 2 minutes in an Eppendorf centrifuge, transfer the upper aqueous phase to a fresh tube. Avoid the material on the interface between phases!
11. Add two volumes of 100% ethanol at room temperature. Mix by vortexing. Stand at room temperature for 2 minutes.
12. Centrifuge for 5 minutes in an microcentrifuge at room temperature.
13. Remove the supernatant. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away.
14. Add 1 ml of 70% ethanol. Vortex briefly and then recentrifuge. THis step is called "washing the pellet". Q: Does the pellet dissolve?
15. Again remove all of the supernatant. Air dry the pellet briefly (5 minutes) after wiping away residual ethanol with a Kimwipe.
16. Add 50 µl of TE (pH 8.0). Vortex and incubate at 37o C to solubilize DNA (about 5 minutes). Vortex several times during heating. Store at -20oC until next lab session.
Feb 16 Restriction analysis of plasmids and Quantitation of DNA
A. Restriction analysis of plasmids To analyze plasmids from overnight cultures, we'll restrict with EcoRI and Xho I to see if the 400bp insert is present.
1. Remove 10 µl of the plasmid solution to a new Eppendorf tube. Add 5 µl, H2O and 2 µl of the appropriate 10X buffer (____) and 12 units of the desired restriction enzyme(s) (Sac I and Xho I. Can use 1 ul each). Add 1 µl 1 mg/ml RNase to each tube. Incubate for 1 h at the appropriate temperature (37° C). Also make one uncut plasmid sample from one of your minipreps: 10 µl + 1 µl 10X dye. Store the remainder of the plasmid preparations at -20 ° C.
2. Make a 1% agarose gel + 0.5 µg/ml EtBr in 1X TAE. Use a mid-size apparatus and two 14-well combs.
3. Add 2 µl 10X DNA dye to @ digest.
4. Load gel with uncut plasmid (1 sample) and 6 plasmid digests. Include a Lambda Hind III EcoR1 markers (1 µg) in one well of top and bottom tiers.
5. Run at 120V, 1 h.
6. Photograph under UV light.
7. By comparing the bands in plasmid lanes with Lambda standards, one can describe
a. The molecular size of the fragment
b. The [DNA] of each fragment, and
c. the molecular form (circular vs. linear) of the fragments
For (a), compare migration distance to that of Lambda standard fragments - estimate bp size
For (b), compare brightness of bands to those of Lambda fragments - estimate ng. Divide by 10 µl (volume of plasmid DNA miniprep in the sample loaded) to get [DNA] in the miniprep. This is often more reliable quantitation than A260 measures!!!
For (c), note that uncut plasmids run fast as two forms, supercoiled and nicked circular. The restricted plasmid from vector only transformation shows that linear DNA is slower. Since markers are linear DNAs, their migration only relates to other linear DNAs.
Q1. Which clone(s) are pbUB-GX2? How do you know?
B. Quantitation of plasmid DNA by Absorbance at 260 nm
Absorbance measures of DNA & RNA at 260 nm are used to estimate concentrations of nucleic acids. An absorbance of 1.0 for solution of double-stranded DNA has = 50 µg/ml while RNA has A260 = 40 µg/ml and single-stranded DNA A260 = 37 µg/ml. An unknown sample of DNA can be measured for A260 and
[DNA] = A260 X dilution factor X 50
The ratio of A260/A280 is an indication of the
purity of the nucleic acid. The ratio for pure DNA is 1.8 while for RNA it is
2.0. Protein, phenol, EtOH and other things interfere
and often lower these ratios because they absorb at A280.
FOR each group:
1. Add 1200 µl of H2O to 8- 1.5 ml tubes.
2. Label one "Blank" and the others (duplicates) after the DNA samples: Your 6 plasmid minipreps. Also make a dilution of a positive control: 10 mg/ml salmon sperm DNA.
3. Add 6 µl aliquots of DNA to @ of the tubes except the "Blank".
4. Measure the A260 & A280. Use a UV-transparent plastic cuvette and transfer pipets.
5. Estimate [DNA] (µg/ml) = A260 · diln. factor · 50
= A260 · 200 · 50
Therefore: [DNA] (mg/ml) = A260 · 10
NOTE: mg/ml = m g/m l
6. Determine the masses of plasmid DNA from gels you have run.
DISCUSS:
2. Compare the mass determinations from gels with those from A260 s. Do they agree? If not, why? (Hint: Look at the uncut control on the last gel).
3. What are the A260/A280 ratios? What are expected ratios for pure DNA and Pure RNA?
Feb 18 Transform a pUB-GX2 clone into JM109(DE3)
YOU WILL BE USING LIVE BACTERIAL CELLS -
DECONTAMINATE WITH BLEACH OR DISCARD IN BIOHAZARD WASTE.
1. From the last agarose gel, each student should choose a pbUB-GX2 clone. Transform it and pGEMEX-2 as you did for the positive control for the first transformation.
2. Determine what the concentrations of the plasmids are from past information obtained. (For the pUB-GX2 clone, should you use the A260 or the gel determination from last lab?
3. Dilute a minimal amount of each plasmid separately to 10 ng/ul with TE buffer
4. Transform JM109(DE3) cells with 10 ng plasmid as you transformed before. Since you are transforming circular plasmid DNA, how many ul of the SOC culture will you spread on the LB-Amp plates?
DISCUSS:
1. What’s the difference between JM109 and JM109(DE3) E coli strains?
2. Why didn’t we use JM109(DE3)
from the beginning
Feb 23 Grow recombinant cells expressing UB protein
YOU WILL BE USING LIVE BACTERIAL CELLS - DECONTAMINATE WITH BLEACH OR
DISCARD IN BIOHAZARD WASTE (will be autoclaved).
A. Grow cells
1. Record observations from plates
2. Each student should pick one pUB-GX2 colony and one pGEMEX colony and inoculate separately into 20 ml LB containing 100 ug/ml ampicillin
3. Grow O/N at 37 °C with agitation (of the cultures, not you).
B. Protein dot blot controls:
This filter will carry three dots of a purified protein that won’t bind the antibody, and three dots of a protein
mixture that includes ubiquitin and therefore should
bind the antibody. These will serve as positive and negative controls for
future immunodetection (they will be incubated with
blocker, antibodies and developed alongside the Western blots).
1. Each pair of students should make a dot blot with Bovine serum albumin (10
mg/ml) and endometrial cytosolic extract. Spot each
undiluted, 1:10 and 1:100 dilutions in phosphate-buffered saline. Use 1 ul volumes on a strip of Hybond
ECL.
Allow the filter to airdry and store at RT until next time.
WATCH VIDEO: PROTEIN GEL ELECTROPHORESIS
TA’s will store the cultures at 4 °C tomorrow
for the next lab.
Feb 25 Inducing the Production of UB Protein and Setting up the PAGE gel apparatus
YOU WILL BE USING LIVE BACTERIAL CELLS - DECONTAMINATE WITH BLEACH OR DISCARD IN BIOHAZARD WASTE.
Each student should continue his/her two cultures.
- Resuspend cells from the overnight cultures by gentle swirling.
- Inoculate 5 mls of O/N culture into 20 ml of fresh LB+Ampicillin (100 ug/ml)
- Grow with shaking at 37 ° C until the absorbance at 600 nm is 0.2 to 0.5. Remember to use sterile technique when you remove an aliquot for absorbance measurement. Discard the aliquot in appropriate biohazard waste.
4. Add IPTG to final concentration of 0.5 mM.
5.Grow O/N with shaking at 37 °C.
B. Setting up the PAGE apparatus- Each pair of students should set up one gel.
During step 3 (above) set up the SDS-PAGE mini-gel apparatus in the gel
pouring stand. .
- Assemble the gel plates and spacers for gel pouring following manufacturer’s or instructors’ instructions. Test the alignment of bottoms of plates and spacers by dragging a thumbnail across both bottom corners. If alignment is perfect, leaks will probably not be a problem.
- Test the setup for leaks by filling it with Nanopure H2O. Drain the water. Store for use in next lab.
Feb 26 TA’s
will store cultures at 4 °
C.
Mar 1 Make
the SDS-PAGE gel and Prep the UB Protein Samples
NOTE: Wear gloves. Unpolymerized acrylamide is a neurotoxin.
A. Pour the Separating Gel:
- When you have a good gel set up (no leaks), pour the SDS-PAGE separating gel.
2. Add the following components in a 250 ml beaker, and swirl gently to mix the separating gel:
Water 6.0 ml
30% Acrylamide 5.0 ml
1.5 M Tris( pH8.8) 3.73 ml
10% SDS 0.15 ml
10% APS 0.2 ml
Q: What percentage acrylamide is this gel?
3. With a Sharpie, mark a
line 2 cm below the top of the short plate. When you are ready to pour gel, add
10 µl TEMED to beaker, swirl gently, and immediately pour solution into gel
mold using a 10 ml graduated pipette. Pour in gel up to the line you drew.
Gently overlay with 1 ml of H2O using a P1000 pipetman.THe
interface between gel solution and water will be invisible until polymerization
occurs.
Watch leftover gel in beaker for
polymerization, which should occur in 10 to 20 min. NOTE: O2 inhibits
polymerization so the surface solution will not polymerize!
B. Prepare the protein samples – Each student should make one sample each for both pUB-GX2 and pGEMEX-2 clonal cultures.
LIVE BACTERIAL CELLS – Discard waste appropriately!!
- Swirl the O/N, IPTG-induced culture to resuspend the cells.
- Pellet 200 ul of cell culture by a brief spin in a 1.5 ml tube.
- Discard supernatant and resuspend cells in 30 ul TE
- Lyse cells by adding 30 ul SDS sample buffer and incubating in a 95 °C block for 5 min.
5. Store samples at -20 °C .
C. Pour the stacking gel
1. Mix solution for stacking gel.
Water 5.6 ml
30% Acrylamide 1.6 ml
0.5 M Tris pH 6.8 2.5 ml
10% SDS 0.1 ml
10% APS 0.1 ml
When you are ready to pour the stacking gel, pour off water overlay on top of the separating gel.
Then add 10 µl TEMED to solution, pour immediately and insert comb. Insert comb so teeth are stuck 1 cm above the separating gel. So the well depth is 1 cm and the functional depth of the stacking gel is 1 cm.. Watch the residual stacking gel to check for polymerization (usually about 15 min).
Store the gel under 1X stacking gel buffer on gel and on a dampened paper towel (leave the comb in) on your bench. Wrap whole apparatus in plastic wrap.
Q.: What are the differences between stacking and separating gels?
Mar 3 Running the SDS-PAGE gels and Western blotting
A. Running the SDS-PAGE
1. Heat prepared samples at 90°C for 3 min. along with prestained
protein markers markers.
2. Prepare 300 mls of 1X gel running buffer (25 mM Tris/250 mM glycine/0.1% SDS)
2. Gel should be loaded left to right:
Lane 1: Prestained protein standard markers from BioRad (see product information for protein sizes)- 5ul (Can watch these during electrophoresis to visualize separation in real time, as well as they provide a visual assessment of the efficiency of protein transfer to blots).
Lanes 2 and 4: 25 ul of pbUB-GX2 culture protein from student #1 (lane 2) and #2 (lane 4)
Lanes 3 and 5: 25 ul of pGEMEX-2 culture protein from student #1 (lane 3) and #2 (lane 5)
Lanes 6-10: Repeat lanes 1 to 5 above.
Use gel loading tips to deliver the sample directly to the bottom of the wells.
(NOTE: Lanes 1 to 5 will be immunoblotted, while 6 to 10 will be stained for total proteins with Coomassie stain.)
5. Run gels at 200 V until bromophenol blue reaches the bottom of the gel, approx 45 min.
6. Turn off power supply. Disconnect electrodes. Remove clamps from sides of gel. Place gel in glass plate sandwich flat on table on top of paper towels. Remove side spacers. Use spacer to pry glass plates apart. Remove tall plate, leaving gel attached to short plate, if possible. (If gel comes off on tall plate it will be backwards its original loading order, that is lane 1 will be at far right) Remove and discard stacking gel.
7. Discard stacking gel. Cut Lanes 6-10 from rest of gel and stain in Coomassie + Fixer with gentle agitation (use a shaking platform) at room temperature in a sealed container (Tupperware) until next lab. (REST OF GEL GETS BLOTTED IN SECTION B).
8. Identify the marker bands and record their migration distances from the top of the separating gel.
B. Electrophoretic transfer of proteins in Lanes 1-5 to nitrocellulose membrane
NOTE: Wear gloves when handling nitrocellulose membrane. Otherwise oil, proteins and nucleic acids from fingers will cause blotches on the filters.
1. Measure size of gel containing Lanes 1-5 in cm. Soak gel in transfer buffer for 15 min at RT.
2. Cut six sheets of 3MM chromatography paper a little bigger than the gel to be blotted. Cut one piece of Hybond ECL nitrocellulose membrane to the exact size act size of the gel to be blotted. Mark the membrane with your initials in two opposite corners with pencil or black ball point pen. Note markings and corners.
3. Wet the Hybond ECL nitrocellulose filter by floating on a pan of distilled water, and then submerge the filter in water for 2 min.
4. Wet the chromatography filter papers by soaking in western transfer buffer (see Fig 5B). Chromatography filter papers should be dripping wet.
5. Assemble the gel 'sandwich' in the order from bottom to top (Fig. 5B). Check carefully for air bubbles at each step and gently remove them using a gloved hand. When stack is complete, roll a pipet over the sandwich to ensure that bubbles are removed. Mark the outer Whatman paper with your initials on the nitorcellulose (not gel) side. This side will be oriented to the anode in #6.
6. After assembly, place the sandwich in transfer unit IN THE CORRECT ORIENTATION!!! and submerged in the transfer buffer. Connect the electrodes. Transfer O/N at 15 V and at 4 ° C with buffer stirring (unit sits on a stir plate)..
Clean up all plates, etc with soap and rinse in H2O and dry. Return
them to their storage cabinet.
A.
B
Figure 5. Schematic of arrangement for electrophoretic transfer of proteins onto a nitrocellulose membrane. Panel A shows the order for setting up the gel 'sandwich' and panel B shows the completed sandwich on the gel unit in proper orientation for electrophoresis. Whatman papers are wet in Tris-glycine transfer buffer.
Mar 8 Blocking and Incubation of Blots with Primary Antibody
GENERAL: Wear
gloves at all times when handling blotting membranes. Note
which side protein is on and do incubations with that side up.
Figure 6.
Detection of proteins immobilized on nylon membrane using a specific primary
antibody and horse radish peroxidase (HRP)-conjugated
secondary antibody.
To identify ubiquitin protein, the blot containing Lanes 1-5 will be developed as an immunoblot: with primary antiserum raised in rabbits against ubiquitin protein as the first reagent. Immunoblots are used to detect small amounts of a particular protein using specific antibodies. We’ve already done much of the procedure: separation of proteins on SDS gels, transfer of proteins to a membrane support. Now we will block of nonspecific binding sites on the membrane and add primary antiserum and incubate overnight at 4 o C.
Next lab, we will do the detection part: binding of a secondary antibody (goat anti-rabbit IgG conjugated with horse-radish peroxidase (HRP)), detection with peracid substrate, luminol, and enhancer. The detection system generates low levels of light at the site of antibody binding.
This is detected on X-ray film.
TODAY:
- After transfer, disconnect the power supply. Dissasemble the blotting apparatus.
- While blot is still on the gel, mark the top and bottom and lanes on the edges of the blot with a sharp pencil . Also label initials so can tell which side of the nitrocellulose that the protein is on.
- Stain blotted gel with Coomassie as in A7 to assess completeness of protein transfer (can put it in same container as gel with lanes 6-10.).
- Place the gel blots on paper towels to let them air dry.
- Place nitrocellulose membranes (Western blot lanes 1-5 and protein dot blot from an earlier lab) in a labeled yellow tip box lid. Add 20 ml blocker to each lid and incubate with agitation at RT for at least 1 h.
- Replace the blocking solution with primary antibody solution and incubate O/N at 4 °C with gentle shaking.
- ALSO:
Destain
the two gel pieces stained with Coomassie and take a
picture of them together (reassemble the gel) on a white light box with
Polaroid 667 film and f-stop 32 or 16 for 1/125 sec. Lanes 6-10 show us
that the E. coli lysates have a lot of proteins in
them. The electroblotted gel (lanes 1 to 5)
should be blank, demonstrating that protein transfer was complete (hopefully).
Q1: Does the induction of UB protein synthesis noticeably change the pattern of total proteins observed with Coomassie staining?
NOTE: Protein sizes can be determined by comparison to the migration of the molecular weight markers. In your spare time during this lab, graph the migration distances of the protein markers against the log of their molecular weights. From this standard curve, determine the molecular weight of one band on the immunoblot. The graph goes in your notebook and the data goes in your report.
· Q2: What is the expected size (relative molecular weight = Mr) of the T7 gene 10-UB fusion protein in daltons?
Mar 10 Immunodetection of UB protein
GENERAL: Wear gloves at all times when handling blotting membranes. Note which side protein is on and do incubations with that side up. Once the washing procedure begins, the blot should be kept wet at all times. Do not allow the blot to dry during changes of buffer. At each buffer change, try to remove all of old buffer before adding fresh buffer. The times given below can be extended, but not shortened.
1. Can wash several Western blots together in a tupperware container with lid (4 groups together):
Wash blots in TBST (Tris-buffered saline plus Tween-20) at RT:
a. Drain filters and discard blocker plus antibody solution (NOTE: This solution can be reused for months!! But you can discard yours.) Rinse the filters twice briefly with 100 ml of TBST
b. Wash once with 200 ml TBST for 15 min, with gentle shaking.
c.Wash twice with 200 ml TBST for 5 min, with gentle shaking.
2. Place each blot in a clean yellow tip box lid:
Add minimal amount (10 ml) secondary antibody solution. Shake gently for 45 min at room temperature. Discard the secondary antibody solution.
3. In tupperware, wash blots together in TBST at RT:
a. Rinse twice briefly with 50 ml of TBST
b. Wash once with 100 ml TBST for 15 min, with gentle shaking.
c. Wash four times with 100 ml TBST for 5 min, with gentle shaking.
4. Wear gloves to protect blot!
a. Combine 2.5 ml detection reagent 1 with 2.5 ml detection reagent 2.
b. Drain off all wash, put blot on a piece of plastic wrap and add 5 ml from step "4a" to blot while holding up the edges of the plastic wrap. Keep blot flat on the counter for a 1 min incubation (EXACT TIME).
c. Drain off excess reagent. Wrap the blot well in plastic wrap (No leaks! Double fold the wrap edges under the blot).
d. Put on X-ray film in a cassette in the dark room using only safelights for light in the room (see instructor for directions). Develop the film after a 5 min exposure at RT. Re-expose the blot to film if necessary.
Mar 22
Introduction to Module 2 – Cloning and Identifying a Gene Product
In this module, each student will work on their own assigned cDNA (reverse-transcribed from a mammalian mRNA). There are no assigned activities on a day to day basis. Instructors will assist students in designing their particular goals for the day and setting up the experiments.
NOTE: Appropriate controls (positive and negative) can make the difference between conclusive and inconclusive results!!!
NOTE2: Exercises may overlap.
The last wet lab is May 3.
Each student will seek to identify the gene product encoded by the mRNA
and to use genetic engineering to direct its expression in a transfected mammalian cell.
Each student will be supplied with a set of nested PCR primers and total cellular RNA from an animal tissue. With PCR, each will amplify a complementary DNA fragment from a messenger RNA. But first, you will have to reverse transcribe the mRNA to cDNA. BE CAREFUL TO AVOID RNase contamination of this reaction….use RNAse free tips and tubes and reagents.
Then PCR with the “outside” set of PCR primers….then PCR with the “inside” set of primers.
Questions to be addressed:
Answer these questions in your notebook!
Q1. WHAT size (in bp) AND HOW MUCH DID DNA
(in ug) did you generate?
To generate a recombinant clone, ligate the PCR
product into a TA cloning vector and amplify the clone as a plasmid DNA.
Confirm presence of insert in at least two independent plasmid clones by restriction
digestions.
Q2. WHAT IS THE GENE PRODUCT ENCODED IN
YOUR cDNA?
Cycle sequencing (with Polymerase chain reaction) . Sequence two independent clones.
Watch Video: Dideoxy Sequencing
reaction during gel run.
IFor accurate sequencing,
the DNA needs to be sequenced on both strands. Do one sequencing reaction with
the "M13 forward" and another with the "M13 reverse"
sequencing primer, each on the same plasmid chosen in A, above.
1. Program the PCR machine for
30 cycles of: 96°C
20 sec
50°C 10 sec
60°C 4 min
2. In a labelled 200 ul thin-walled PCR tube, combine:
2 ul Big-Dye mix* (Applied Biosystems product)
___ul DNA template (400 ng)
___ul PCR primer (10 pmol; "M13 forward" or "M13 reverse")
___ul ddH20 (to final volume 7 ul)
3. Mix well and place in a thermal cycler
4. Run the PCR
5. Either set thermal cycler
in a cold room so samples will be held at 4°C
or retrieve tubes and store at 4 °C tomorrow.
*Q.: What’s in the mix?
Removal of Unincorporated Nucleotides from PCR sequencing Reactions and submitting for automated sequencing: Beginning Manual sequencing with Boehringer Mannheim’s DIG (digoxigenin) Taq DNA sequencing kit.
A.To separate the labeled DNA from unincorporated labeled nucleotides, a spin column is used. These can be made, as described below, or purchased. Centrifugation forces the fluid through the gel matrix of the column. The small nucleotides get hung up in the beads, while the large DNAs fall through and are collected in a tube below the column.
1. Get a Bio-Rad Micro Bio-Spin Chromatography column and collection tube (2 ml).
2. Invert the column sharply several times to resuspend settled gel and remove bubbles. Snap off the tip and place column in 2 ml tube. Now remove top cap. Allow buffer to drain by gravity and discard it.
3. Centrifuge for 2 minutes in a tabletop IEC centrifuge at 2500 rpm and discard buffer.
4. Increase the PCR volume to 50 ul with water. Load the PCR sequencing reaction in a 50 ul volume on the center of the gel column with a clean tube (1.5 ml) as collection tube.
5. Centrifuge for 4 min at 2500 rpm. You may have to cut the tube cap off so label the tube body.
6. Open the tube cap. Wrap the tube opening with Parafilm. Stab the Parafilm with
a needle to create holes. These samples will be speed-vacuum dried, then
submitted to Biology Gene Technologies core lab for running on an automated DNA
sequencer.
Watch VIDEO: DNA Sequencing Electrophoresis
Q3. WHAT is the. IDENTITY of THE
GENE YOUR cDNA COMES FROM?
Sequence analysis and similarity searches in GenBank
In this lab we'll identify a protein product encoded by the cDNA in your plasmid and believed to be related to that in pUNKNOWN. Choose one sequence, from either automated or manual sequencing results. After removing the vector sequence (only enter the sequences following the EcoR I restriction enzyme site "GAATTC"), search at least 50 bp of cDNA sequence for similar sequences in GenBank with the BLAST software at the Genbank website (http://www.ncbi.nlm.nih.gov/Genbank/GenbankSearch.html). Choose "BLAST Sequence similarity searching." Choose "Basic BLAST search." The program is "blastn" and database to be searched is "nr" the nonredundant nucleotide database. Enter the sequences of the cDNA in "FASTA Format": the first line can contain a name of the sequence and other info preceded by a ">" . Hit "enter" and start entering sequence only on the next line. Make sure you enter the sequence in the 5’ to 3’ direction. If you do not, the reverse sequence actually bears no relation to the correct one except for having the same overall nucleotide compostition. If you are working during the weekday, sometimes the search takes a while. You can e-mail the results to yourself to save yourself from a long wait. Or you can wait and get the results from the website. Sample BLAST results are:
<b>BLASTN 2.0.3
[Nov-14-1997]</b>
<b>Reference:</b> Altschul, Stephen F., Thomas L. Madden, Alejandro A.
Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman
(1997), "Gapped BLAST and
PSI-BLAST: a new generation of protein database search
programs", Nucleic Acids Res.
25:3389-3402.