I am looking for help to optimize a nested PCR from Long Range (LR) PCR (DNA as template). The LR PCR product looks spesific on agarose gel. We have tried various dilutions of this product as template, but keep getting a couple of unspesific bands on the gel in addition to the expected product (the unspesific bands are a little larger in size than the expected product). When we add genomic DNA instead of the diluted LR PCR product, using the same polymerase and conditions, we get clean product of the correct size. What could be the cause of the unspesific bands? Is it necessary to clean up the LR PCR product before using it as template for nested PCR when we dilute it (1:50, 1:100, 1:1000)?
over amplification is a problem in nested pcr. So every 10 cycles is 1000 times more pcr product. I would use the first round pcr product at 1/1000 dilution and make up 8 tubes for pcr then set to 30 cycles and remove tubes at 12,14,16 etc cycles then at some cycle number you should get strong product amplification but weak non specific amplification. If theis is still a problem try 6%dmso in the pcr mixture or hemi nesting...outerf-innerR and innerF to outer R if you just need a product
Thank you so much for your feedback! We will try the various number of cycles first like you suggest.
The unspesific bands are actually a little smaller than the correct product, not larger as I wrote initially, but I assume the advice is still the same?
Smaller is better because it eliminates the possibility that the extra band (if larger) is a heteroduplex of mismatched bases in the amplimer forming a bubble and the product appears to run slower (larger) than the homoduplex and these tend to reform whatever you do in the way of purification because your pcr product may have been 2 very similar sequences (one base changed but no difference in length)
Hi, additionally to upper comments, you may treat your 1st step product after LR PCR with ExoSap/cut from gel for eliminate residue of primers from 1st step (outer primers) and dilute treated product for 2nd step, thus you can avoid non-specific product which can produce if you have residue primers from 1st step.
In my experience - ExoSap/cut from gel treat after each step of nestedPCR provide easily calibration for cycles on next step and lower numbers of non-specific bands.
Hope it's helpful for you :)
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