Sequencing of the cloned plasmid
Posted 22 July 2013 - 01:03 AM
I cloned a 3.2kbp of fragment into pGL3-basic vector and then sent to a company for sequencing with RV3 and GL2 primers.
The sequencing results with RV3 was good, but with GL2 had lots of noise and I could perform alignment.
Do you know what could cause that problem?
Thank you very much for helping me out.
Posted 22 July 2013 - 07:08 AM
Posted 22 July 2013 - 05:27 PM
Thanks for your suggestion.
However, if I got 2 different plasmids in one colony, or 2 inserts in one plasmid, the noise should happen with both GL2 and RV3 primers, but it only happened with GL2.
Moreover, I used gel electrophoresis with non-cut and cut plasmid with specific RE to confirm the insert, the bands were totally right in size of insert and plasmid.
So, I think the problem should be due to the contaminated of the GL2 primer...
Do you think so?
Posted 22 July 2013 - 08:20 PM
Not necessarily, as you are sequencing a big insert, the noise could quite easily be seen only on one end if you had more than one insert in the same plasmid. Two different inserts would probably give you noise at both ends, but not if they shared an identical start. Splice variants would be one form of a gene that could give this result.
However, if I got 2 different plasmids in one colony, or 2 inserts in one plasmid, the noise should happen with both GL2 and RV3 primers,
Cutting out the band won't tell you if you have back to back insertions as they would be digested between the two inserts.