20.109(S13):PCR and paper discussion (Day3): Difference between revisions
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including why we are using a proofreading polymerase and BSA in this particular situation | including why we are using a proofreading polymerase and BSA in this particular situation | ||
We can improve the reliability and accuracy of PCR, especially important when the DNA originates from a stool sample, by two key methods. The first is the use of a highly specific polymerase, one with either explicit or inherent hot start properties. Hot start means that the polymerase is inactive at low temperature <font color=red>(add some further elaboration here)</font color>, while the reaction is being set up and initially heated, thus reducing non-specific interactions between primers and non-target DNA. The important point to note here is that the target DNA (DNA of interest) may be present at a low concentration compared to the DNA in the sample as a whole, increasing opportunities for non-specific binding. The second performance enhancer is adding bovine serum albumin (BSA) to the PCR. As you saw if you clicked on the Kreader paper linked on Day 2, BSA itself binds many inhibitors of PCR, thus acting as a competitor with respect to the polymerase. We would much rather that inhibitors bind the BSA than bind the polymerase and interfere with its function! BSA is hydrophobic and somewhat positively charged, making it a great non-specific binder of proteins that we will use time and again in 20.109. | |||
Even taking the above precautions, you might expect to obtain some non-specific products in your PCR today. Next time, you will use a gel to both visualize the PCR (potentially multiple bands) and to purify the band of interest. | |||
maybe gel electrophoresis background also, so don't have to cover it along with everything else next time | maybe gel electrophoresis background also, so don't have to cover it along with everything else next time | ||
Revision as of 14:38, 7 January 2013
Introduction
PCR background
including why we are using a proofreading polymerase and BSA in this particular situation
We can improve the reliability and accuracy of PCR, especially important when the DNA originates from a stool sample, by two key methods. The first is the use of a highly specific polymerase, one with either explicit or inherent hot start properties. Hot start means that the polymerase is inactive at low temperature (add some further elaboration here), while the reaction is being set up and initially heated, thus reducing non-specific interactions between primers and non-target DNA. The important point to note here is that the target DNA (DNA of interest) may be present at a low concentration compared to the DNA in the sample as a whole, increasing opportunities for non-specific binding. The second performance enhancer is adding bovine serum albumin (BSA) to the PCR. As you saw if you clicked on the Kreader paper linked on Day 2, BSA itself binds many inhibitors of PCR, thus acting as a competitor with respect to the polymerase. We would much rather that inhibitors bind the BSA than bind the polymerase and interfere with its function! BSA is hydrophobic and somewhat positively charged, making it a great non-specific binder of proteins that we will use time and again in 20.109.
Even taking the above precautions, you might expect to obtain some non-specific products in your PCR today. Next time, you will use a gel to both visualize the PCR (potentially multiple bands) and to purify the band of interest.
maybe gel electrophoresis background also, so don't have to cover it along with everything else next time
Protocols
Part 1: Prepare PCR to detect bacterial 16S
need to do microsporidia PCR on separate day because the Ta etc. is different --> need to find notes on where I planned to put that. possibly Day 2? primers should have arrived by then. then intro structure also needs to change
- Begin by carefully labeling each PCR tube that you will use with the date, sample name, and a unique symbol and/or color for quick identification. Filling in the cap tab with your team color will usually suffice. Pre-chill the tubes on a cold block.
- You will now prepare a so-called "master mix," which contains every PCR ingredient except the template and the polymerase. Prepare enough for the number of reactions you need to run, plus an additional 10%. Feel free to use the table below for your calculations. [DOING MORE THAN ONE OR NOT?]
- When the master mix is not in use, keep it on ice.
- Combine 5 μL of template, 45 μL of master mix, and 1 μL of PfuUltra polymerase in a PCR tube. When everyone's reactions are ready, they will undergo the cycling conditions listed below.
| Reagent | Amount for 1 reaction (μL) | Amount for ? reactions + 10% |
|---|---|---|
| PfuUltra buffer (10X stock) | ||
| Primer mix [TO WHAT EXTENT PREPARED FOR THEM?] | ||
| dNTPs | ||
| 10% BSA (100X stock) | ||
| Water | ||
| DNA template | 5 | N/A |
| Segment | Cycles | Temperature (° C) | Time |
|---|---|---|---|
| 1 | 1 | 95 | 5 min |
| 2-4 | 25 | 95 | 1 min |
| 51 | 1 min | ||
| 72 | 2 min | ||
| 5 | 1 | 72 | 10 min |
| 6 | 1 | 4 | indefinite |
Part 2: WAC Session
Today you will hear a lecture on preparing your journal club presentations.
Part 3: Journal article discussion
Scientific papers are dense and often time-consuming to read and understand, but with practice, you will find strategies that improve your comprehension efficiency. Here's one tip to get you started: when reading newly reported results, be sure to refer to the associated figures frequently, because visual information is often easier to take in than purely verbal descriptions.
Technical Background
Discussion Topics
Writing
As you read the paper by WHOEVER, consider not only its scientific content, but also the authors' writing style (perhaps not all on one read!). Sketch out answers to the questions below (right on the paper if you wish). Your answers will not be collected, but you may be called on in discussion to share your ideas.
Content
Probably changing to assigning these in advance and doing slightly more formal presentation (single slide prepared)
When you arrive in lab today, each group will be assigned one of the following topics to present to and discuss with the rest of the class. You should be somewhat familiar with the whole WHOEVER paper by now, but will have some time in-class to refresh your memory and become the resident expert in one of the following areas.
For next time
Reagent list
write something here or not accessible to edit
