I am doing PCR work for the first time. Any tips/suggestions?
Hello Sir,
Before the PCR product is used in further applications, it has to be checked if :
There is a product formed:
Though biochemistry is an exact science, not every PCR is successful. There is for example a possibility that the quality of the DNA is poor, that one of the primers doesn't fit, or that there is too much starting template
The product is of the right size:
It is possible that there is a product, for example a band of 500 bases, but the expected gene should be 1800 bases long. In that case, one of the primers probably fits on a part of the gene closer to the other primer. It is also possible that both primers fit on a totally different gene.
Only one band is formed:
As in the description above, it is possible that the primers fit on the desired locations, and also on other locations. In that case, you can have different bands in one lane on a gel.
Hi.
Are you planning conventional end-pooint PCR or quantitative real time PCR?
Hello Sir,
Before the PCR product is used in further applications, it has to be checked if :
There is a product formed:
Though biochemistry is an exact science, not every PCR is successful. There is for example a possibility that the quality of the DNA is poor, that one of the primers doesn't fit, or that there is too much starting template
The product is of the right size:
It is possible that there is a product, for example a band of 500 bases, but the expected gene should be 1800 bases long. In that case, one of the primers probably fits on a part of the gene closer to the other primer. It is also possible that both primers fit on a totally different gene.
Only one band is formed:
As in the description above, it is possible that the primers fit on the desired locations, and also on other locations. In that case, you can have different bands in one lane on a gel.
My first advice is to run each sample in triplicate for each gene. this may seem a waist of reagent and space on the plate, but at the end it will give tighter Ct values.
If you want to save master mix and primers you can scale down the final volume of your PCR to 10-20 microliters/well.
My second advice is to run a calibration curve for your genes of interest and housekeeping: start from a certtain amount of cDNA (e.g., 50 ng/microliter), make serial dilutions (usually 1:2 or 1:10), and add equal amounts (2-5 ul) of serially diluted cDNA/well. Run the qPCR, plot Ct value vs therotical amount of cDNA, and make a linera regression. The more steep is the linear regression (ideally -2.3) the more efficient is your qPCR for that particular gene. If the qPCR has the same efficiency for all the genes you are analyzing using the same amount of cDNA, you are fine. Otherwise, you must optimize the amount of cDNA and make sure that the efficiency is comparable/acceptable.
If you are using SybrGreen as fluorescent dye, run a dissociation curve at the end of the qPCR thermal cycles. Thi swill help you to identify erroneous amplification by simply looking at the melting curves.
Hope this will help, but feel free to contact me again.
Good luck with your qPCR
Antonio
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