Expression Vector sequencing problem - (Jul/08/2008 )
I have a very strange problem.
I have solved most of my problems thanks to Protocols Online. I was one of those people who had a hard time with cloning. But after my 22nd ligation, I have 3 positive clones! I was happy into sending them for sequencing and the sequence is as I want it and is in frame.
I went up happily to tell my supervisor when she told asked me to check the vector itself.
I am using the pQE30 expression vector and am using SphI and HindIII as my cloning site.
The Met and 6x His Tag is fine but at the beginning of the sequence there is a 13 nucleotide frameshift. Is this normal? Can I still use my positive clone for expression work? Or do I need to purchase a new stockof pQE30. Or is it the sequencing problem?
My methods in a dash : -
I cut my 7ug of vector with 30U of SphI and HindIII overnight. Purify with Qiagens Gel Extraction kit. Ligate overnight with 100U of T4 DNA ligase by NEB. Plate on LB-Amp and use LB-Amp broth for screening. I purify my samples with SV minipreps (Promega, USA) and send about 15uL (80ng/uL) with 50pmol of the PQE forward primer.
CTCGAGAAAT CATAAAAAAT TTATTTGCTT TGTGAGCGGA TAACAATTAT AATAGATTCAATTGTGAGCG GATAACAATT TCACACAGAA TTCATTAAAG AGGAGAAATT AACTATGAGAGGATCGCATC ACCATCACCA TCACGGATCC GCATGCGAGC TCGGTACCCC
I badly need help. Thank you.
does this frameshift means anything ? I mean a flag or epitope, anything relevant ?
If you feel your insert is ok, go for sequencing the vector used to determine if the original backbone is ok.
Check also your primer sequence.
I see a 140 bases match on these 2 fragments.
where is the 5' of the sequence you obtained from sequencing facility ?
Do you have also the reverse sequence to cross your findings ?