You are asked to amplify a 5kb segment of DNA from double stranded genomic DNA (

Question

You are asked to amplify a 5kb segment of DNA from double stranded genomic DNA (the template DNA). You choose to use Taq Polymerase to amplify your fragment and you are given primers with a melting temperature (Tm) of 65C. You used the thermocycler parameters below to complete 30 rounds of PCR, but you did NOT see a product when you analyzed your PCR sample by agarose gel electrophoresis. In order to optimize your PCR reaction to get your amplified DNA, list two things you would change about your thermocycler conditions and why/how these changes would affect the PCR outcome. Assume 30 cycles is sufficient to see your desired product, the denaturation and extension temperatures are correct, and the initial denaturation and final extension are correct. (2 pts)

PCR Thermocycler Conditions

initial denaturation: 94C for 2min template denaturation: 94C for 20sec

primer annealing: 68C for 20sec primer extension: 72C for 1min

final extension: 72C for 5 min

2) What is the acceptable temperature range for primer melting temperature (Tm)?

3) What is the acceptable percentage range for GC content of primers?

Explanation / Answer

1) Probably there was no PCR product because the primer was undergoing secondary annealing because of higher melting temperature. Therefore less annealing time followed by extension at low temperatures such as 60- 65 degrees would probably amplify the DNA.

2) Primers with melting temperaure range in 52-58 degree celcius usually produces good results.

3) The acceptable GC content range is 40-60%.

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