I did cloning with pGEM-T kit and transformed into a DH5a cells. I've already grown the white colonies overnight and decided to check the insert before going for plasmid extraction and sequencing. I did colony PCR using primers targeting the vector DNA flanking the insert and I got good amplification (~700bp; my insert is ~550). However, when I did colony PCR on the same samples using insert-specific primers, very few samples had amplification of the right size. So I know the samples which had amplification with insert-specific primers are reliable samples for plasmid extraction and sequencing, but should I discard those that had amplification with vector-primers but not with insert-primers?
Hi! I would go with only the ones that were positive when using gene specific primers. Do not try the combination of vector and gene specific primers, because in TA cloning, you don't know the orientation of your insert inside a vector. Maybe your vector forward and insert reverse primers are pointing to the same direction
Yes. You just need one +ve clone. So I would suggest to go ahead with clones which showed desired amplification with gene specific primers may be just for a trial you can select one sample showing 700bp with vector primer. Otherwise what you can do is use forward primer from gene-specific primer and reverse from vector primer in same PCR reaction, if the gene is really intact with vector it should give amplification otherwise no band should appear so no false +ves. Hope it helps.
Hi! I would go with only the ones that were positive when using gene specific primers. Do not try the combination of vector and gene specific primers, because in TA cloning, you don't know the orientation of your insert inside a vector. Maybe your vector forward and insert reverse primers are pointing to the same direction
- Badges
- Science method