User:Anthony Salvagno/Notebook/Research/2009/10/28/Reading about PCR
I decided to read up on PCR to figure out how I can optimize and troubleshoot reactions better in the future. All of my knowledge is coming from Molecular Cloning by Sambrook and Russell.
dNTPs
While I knew that they were important because they make up the backbone of DNA, I did not know the following facts:
- too much is bad in the sense that it is inhibitory "possibly due to the sequestering of Mg2+"
- aliquot stocks of dNTP because they go bad quickly
- should be discarded after 2 cycles of freezing/thawing - Yikes! (Steve Koch 02:25, 29 October 2009 (EDT): Surprising to me too. "Molecular Cloning" kicks ass.)
(Steve Koch 02:27, 29 October 2009 (EDT): Also, just something while I'm thinking about it: It's possible in some conditions to run out of dNTPs or primers! You usually think about both of them sort of being constant concentration. But, for example, if you're doing very short PCR, you could deplete your primers quite a bit if you want a lot of product. Something to keep in the back of your mind.)
Divalent Cations
Whose it, whats out? Apparently this is the Mg2+ stuff. Here is what I read in summary:
- Needed for your polymerase (Taq in my case)
- 1.5mM is routinely used, but up to 5mM may work. 4.5mM or more is enough to cause nonspecific priming in some cases.
- If time permits optimization of Mg2+ could be performed by comparing yields of different reactions with .5mM - 5mM magnesium (in 0.5mM increments).
- Too much EDTA or other chelating agents will sequester your divalent guys.
Temperatures
We want to calculate temps for our primers so that we know what temps to use during the cycling. I found a simple formula:
Tm = 2(A+T)+4(G+C)
The temps for both our primers are 60C according to that equation (dig molecule not considered). For denaturation phase we use 94-95C because that is the most that Taq can handle. For annealing we should use a temp that is about 5C less than Tm. For extension we use 72C which is the optimal temperature of Taq polymerizing.
Times
According to the authors, 30 sec for both denaturing and annealing are good amounts of times. If you have longer oligos more time is needed. We don't, so this is good for us too. The extension time can vary depending on how much template DNA you are copying. A good rule of thumb is about 1kb/min for Taq to synthesize.
Experiment Control
There is a table in the book that I don't quite understand. It looks like this: {{#widget:Google Spreadsheet |key=txqtFh_IuC02n_vytZuLaCw |width=600 |height=300 }}
- Bystander DNA - no target sequences but resembles template DNA in all other respects
- Template DNA - DNA under test
- Target DNA - contains target sequence
Please explain this table to me. Does (+) mean it is in the sample and - means not in the sample?
Troubleshooting
Here is what the book says about my problem of weak/nondetectable amplification:
- Defective Reagent; defective themal cycler; programming error - compare the yields obtained from fresh and old reagents in PCRs incubated in 2 different cyclers
- suboptimal annealing conditions - recalculate Tm; use touchdown PCR with hot start; verify concentration of primers; if primers are culprit make new primers
- suboptimal extension - optimize concentration of MgCl2, template DNA, and dNTPs; test range of pH values in PCR; use fresh Taq; repurify template DNA; increase number of cycles at constant annealing temp; add enhancer if problem persists; if problem still persists reamplify in 1:100 dilution of PCR in fresh PCR stuff or do nested PCR
- ineffective denaturation - increase time/temp of denature
- distance between primers too large - use preps of thermostable polymerases capable of long segments of DNA amplification
From the looks of it numbers 1, 2, or 3 could be the problem. We have tried troubleshooting 3 with some of the suggestions. 1 will be tested tomorrow via thermocouple.
