Hi everyone,
I am currently running a multiplex PCR, and product size varies from 120 bp to 500 bp. I have checked all those temperature on thermo machine, which are fine according to the paper I am following. But it seems all failed. I have run 9 samples, which are all E. coli, the ladder were showing fine, but all those samples showed no "product size" band, while there was a extra small band ( less than 100 bp) appearing for all those 9 samples. PS, cause some products sizes are quite close to each others , so I run the gel slow and long, like 70v: 45 min. Can I anyone please explain this to me?
Hi Huijun,
Are you using a hot start taq? I have seen something similar when trying to perform multiplexed reactions using non hot start taq. The small product generated was primer multimere. With many primers primer/primer interactions can happen readily during reaction set up and (even on ice) can be extended by the taq to produce artifacts that will then interact and prime at the cycle annealing temperature . These primer multimers (these are often referred to as primer dimers, but in reality are often more complex ). can then (because they are short) be preferentially amplified in subsequent cycling.
the extra small band may be the primer multimere, maybe you can do as followings: perform the first PCR using the pair of Forward and Reversed primers, recycle your aimed amplicons, and then you use the second pair of Forward and Reversed primers by using the recycled amplicons as template.
Thank you, Frank and Haihua. I have just run another one this morning, turned out they looked fine for me.
Hello Huijun,
I agree with Haihua. The band below might be primer dimer or PCR byproducts. It is also possible that the annealing temp in your case is different from your reference, so I guess some optimizing is needed. However I see that you have good results, so congrats!
Regards,
NRB
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