This a ligation PCR, which I'm doing it to check if my ligation worked or not. I am using two primer pairs with very similar annealing temperatures. Ideally, if the ligation worked it should answer for both primer pairs. The expected size for the first primer pair is 387bp and the second is 1kb. The template used for the PCR was the different ligation mixes. But both primers had all the ligation mixes. For example, a 1:3 ligation mix was used as the template for both primer pairs. I have thin bands around 0.5kb for the first primer pair indicating the ligation may have worked. But I have no bands for the second primer pair. I am not sure why this is the case. I can only think of two reasons: the template concentration was not enough (cause after all, the second primer is a longer amplicon) or the time I ran was not enough. I had put 45 sec for annealing, assuming that it takes 30 sec for 1000bp, so I should be good anyways. I'd appreciate any help trying to understand what happened! Thank you!
How long was the extension step? It's the length of the extension step, not the annealing step, that has to increase proportionally with the size of the amplicon. I usually give it 1 minute per kilobase to be safe.
I would try repeating the PCR with increased template amount and increased extension time, and if you still see no band, then it sounds like the ligation may not have worked. Do you have a positive control for the PCR?
Some samples may contain inhibitors that can prevent amplification. To determine if inhibitors are present, you can try adding a negative control to the reaction.
Denaturation is at 95°C for 30 seconds. The temperature is then lowered to a temperature that is specific for the primers. Raise to a temperature that is optimal for the DNA polymerase performed at 72°C for 30 seconds.The denaturation, annealing, and extension steps are repeated for 20-30 cycles. Each cycle doubles the amount of DNA that is present in the reaction. The reaction is then held at a final extension temperature for 5-10 minutes.
Cool the reaction and analyze. Incubate the ligation reaction for the correct amount of time, overnight.
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