I am struggling to get a high quantity of DNA after I perform PRC on my sample. I was wondering if anyone could suggest a good PRC reaction (temperatures and times) for a fragment of that size.
PCR optimization is not what can be proposed without knowing particular primers used. Usually temperature varies only at primer annealing steps and is roughly 4(G+C)+2(A+T), where letters are numbers of respective base in the primer (but as a rule exact temperatures are provided by the manufacturer). If primers have different temperatures, the lower one should be selected.
The common way is to optimize your particular reaction with test runs using varying conditions. A good starting point is a protocol from user's manual of used DNA-polymerase. Or, you can look for the protocol in research papers doing similar PCR. Then, perform series of PCR varying following parameters one at a time:
1. Primer concentration (for example, from the initial to the double)
2. Primer annealing temperature. The best is if your cycler supports gradient temperature, this will allow to run PCR with a range of temperature simultaneously. A reasonable range is about +/- 5-6 C from calculated temperature of the primer.
3. Magnesium salt concentration, if it is provided separately from the reaction buffer.
4. Times of reaction steps. However, for 200 bp fragment default values should be OK.
and other tricks, which you can find in literature.
In general, setting PCR for the particular application and in the particular environment is a matter of tries and mistakes and requires some experience.
PCR optimization is not what can be proposed without knowing particular primers used. Usually temperature varies only at primer annealing steps and is roughly 4(G+C)+2(A+T), where letters are numbers of respective base in the primer (but as a rule exact temperatures are provided by the manufacturer). If primers have different temperatures, the lower one should be selected.
The common way is to optimize your particular reaction with test runs using varying conditions. A good starting point is a protocol from user's manual of used DNA-polymerase. Or, you can look for the protocol in research papers doing similar PCR. Then, perform series of PCR varying following parameters one at a time:
1. Primer concentration (for example, from the initial to the double)
2. Primer annealing temperature. The best is if your cycler supports gradient temperature, this will allow to run PCR with a range of temperature simultaneously. A reasonable range is about +/- 5-6 C from calculated temperature of the primer.
3. Magnesium salt concentration, if it is provided separately from the reaction buffer.
4. Times of reaction steps. However, for 200 bp fragment default values should be OK.
and other tricks, which you can find in literature.
In general, setting PCR for the particular application and in the particular environment is a matter of tries and mistakes and requires some experience.
What type of DNA? Is it insect, mammal, bacteria, pant, virus? For mammals you might consider a mitochondrial gene region, you can probably get a small sample of primers from another researcher at you school to test.
Hi,
I always recommend this Paper for PCR optimization:
Article Multiplex PCR: Critical Parameters and Step-by-Step Protocol
Hello, the DNA extraction is from what tissue are you doing it?
You have to check the extraction kit, because many times they vary according to the type of sample, likewise, checking the primers you are using, melting temperature and the small size of the amplicon reduces the time of denaturation and extension.
Roy Rodriguez-Hernandez
Hello,
I am extracting DNA from blood samples. I will check the melting temperature and that stuff. Thank you.
Article A rapid and efficient DNA extraction protocol from fresh and...
To start with you can use gradient PCR with a temperature range of 10 degree depending upon melting points of primers i.e Tm value (written on leaflet of manufacturer). Condition for reaction i.e time depends on which master mix or enzyme you are using (written on leaflets of manufacturer).
I agree with previous answers, primers content and DNA type and concentration are very important
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