We recently have been moving away from using agarose gels and onto using the Tapestation (D1000 for PCR products). However, we have noticed that there are a lot of extra bands that show up on the Tapestation that never showed up on the gel (and been running these in parallel to validate the use of the Tapestation in the lab).
We are running PCR products for Sanger Seq. But every PCR we have done (and with different primers) always show extra bands using the Tapestation compared to a gel. I am aware this is likely a sensitivity issues and that we just couldn’t visualise these bands before on the gel but what are like likely to be? Other PCR products which maybe amplified to lower amounts which is normal?
A primer blast of our primers do not show anything for the bands we see at ~700 bp (product of interest should be around 419 bp but there is sometimes a double band around 300 bp too). Even if the gel was run longer (see attached PDF) we only ever visualise 1 or 2 bands within this 450-300 bp range. So what are the heavier products likely to be?
Thanks in advance!
I am sure that you are right about sensisitivity.Tapestream will detect 5 pg amounts of product invisible on agarose. my feeling is that you will not find out what the other bands are unless you sequence them. Other members of gene families and pseudogenes are always easy to blame for wrong size bands but in the past I have often found that the accuracy of pcr is largely determined by one highly specific primer and the other primer not so good. In this situation it is occasionally educational to run a "PCR" with only one primer and it is surprising how often this produces amplified bands. Unsurprisingly these products sequence badly.
Unspecific amplifications are always difficult to identify. If they don’t interfere with the Sanger sequencing, they shouldn’t be a problem. Do you see the ~700 bp sequence in the electropherograms? You can always increase annealing temperatures and decrease template DNA and number of cycles in PCR to reduce unspecific amplifications. If possible, run a PCR combining each of the primers you are using with alternatives. You may detect the primer causing the unspecific amplification.
- Badges
- Science method