extra band of PCR products by electrophoresis
Extra band of 1st PCR product
Please check the sequence, Tm value of 1st PCR primers and reconstruct if needed.
In 2-step cycle PCR, most of these problems occur due to primer design problems.
If there is a large difference between the Tm value of the forward primer and the reverse primer, please make it Tm value closer.
Please use the 1st PCR Primer design tool in the download section. Each Tm value can be calculated when changing the margin part from 18 mer to 30 mer. From these, primers with a small difference in Tm value can be selected.
Extra band of 2nd PCR product
Please try following
- Change annealing temp from 60 °C to 55 °C ( See Seg.2-2 of protocol )
- Reduce extension time (See Seg. 2-3 and Seg.3-2 )