Splicing DNA fragments - (Mar/09/2016 )

Hi all

I sliced three DNA fragments by overlap extension PCR for generating a construct for knockout. In the sequenced product, everything is fine except that a stretch of about 250 bp is missing as depicted below.

Has anyone ever encountered this? What could be the reason? I visually analyzed the deleted sequence and region flanking it and did not find any repetitive sequence which can cause slippage. Phusion polymerase was used for all the PCRs

Thanks

I never encounter this situation before, did you check your template of the 5' homology region before?

http://www.protocol-online.org/biology-forums/posts/40245.html