After ligating the insert in TA vector pcr 2.1, transformation and miniprep. I amplify the gene with simple gene specific primers . Now I want to sequence the insert . Which primers would I need for sequencing?
you should sequence using plasmid primers mainly because sanger sequencibg will not give good readable sequence under the primer or for the next 40 bases from each end so the sequencing primers should be distant from the insert. this will also give the orientation of the insert and also avoid sequencing post pcr contamination which will amplify gene product even when it is not inserted into the vector
If your insert is less than ~700bp, you could use your primer for seq.
the Reverse primer would get the 5' of your GOI and the forward primer would get the 3' of your GOI if the seq get >700 bp reading.
You could, but it won't give you the orientation of your insert or show if there are any changes right at the insertion site.
I'd use primers from the plasmid that flank the insert. Those are well-designed and are known to give good sequencing results. Plus, you'll be able to tell if you have an empty vector (can be common if you did your screening with colony PCR).
Good luck!
Qurat Ul Ain Ali It is always better to use vector specific primer to get the good quality read of your insert and also the orientation, as suggested by Paul Rutland and others.
The GSPs can be used when you have not cloned your PCR product, however when you have your insert in the vector it is always recommended to go with vector specific primers.
- Badges
- Science method