3 replies to this topic

[seanspotatobusiness]

According to www.dnaseq.co.uk:

Quote

PCR products must be cleaned up to remove unwanted PCR products, excess PCR primers and remaining dNTPs from the PCR reaction. Failure to do this is very likely to result in a messy sequence for reasons which should be obvious, but if they are not, check out our trouble shooting section.

The troubleshooting section does not explain the removal of PCR primers - can't the PCR primers be the same as the sequencing primers? In which case it wouldn't matter whether they were there or not?

[Curtis]

but if you get primer-dimer in your reaction it might interfere with the reading. Better remove them.

[phage434]

If you are left with one primer, you are likely to be left with two.  If you have two, then you will get mixed sequence from the pair of primers, rather than clean sequence from a single primer.  Also, short primer-dimers can vastly outnumber correct bands, yet be nearly invisible on a gel, because they are so short.  Cleanup of your pcr product will tend to remove those, as well.

[seanspotatobusiness]

phage434, on 19 October 2010 - 10:05 AM, said:

If you are left with one primer, you are likely to be left with two.  If you have two, then you will get mixed sequence from the pair of primers, rather than clean sequence from a single primer.  Also, short primer-dimers can vastly outnumber correct bands, yet be nearly invisible on a gel, because they are so short.  Cleanup of your pcr product will tend to remove those, as well.

Ahh, right, yeah, that should be obvious - I wasn't thinking it through. Thanks a lot