Hi,
I have a plasmid, and I want to replace the gene in it with my GOI,
The restriction enzyme site is XbaI(upstream) and BbsI(downstream)
I can design Forward primer of my GOI like this:
F : 5’- ccctcg TCTAGA 111 222 333 444 555 666 -3’
*111 = Start codon of GOI; 222 = 2nd codon of GOI; etc.
But I am really confused how to design Reverse primer for BbsI
The sequence around BbsI site on the vector is
5’… GGTTGAGGTCTCTAAAAGC GTCTTC CT … 3’
3’… CCAACTCCAGAGATTTTCG CAGAAG GA … 5’
This enzyme site is really tricky, does anyone have a suggestion to design the reverse primer?
Hi, Stefano!
You are so nice, your reply is just like a detail protocol! thank you!
I have looked into the BbsI site on NEB. But it is different from yours, it is:
5'- GAAGAC(N2) -3'
3'- CTTCTG(N6) -5'
I mean, the (N2) is flank in the right site of GAAGAC, not left. Similaly, (N6) in the right of CTTCTG.
vector backbone is the following:
5’… GGTTGAGGTCTCT^AAAA GC GTCTTC CT… 3’
3’… CCAACTCCAGAGA TTTT^CG CAGAAG GA … 5’
So, after insert my GOI, is that will be:
5’… 20-mer^AAAA GC GTCTTC CT… 3’
3’… 20-mer TTTT^CG CAGAAG GA … 5’
So, Rev Primer will be:
5'- GAAGAC GC TTTT 20-mer -3'
Furthermore, do you think it's necessary to add flanking site(xx xxx) in front? like:
5'- xx xxx GAAGAC GC TTTT 20-mer -3'
Since the cut site is not in the edge(XbaI T^CTAGA). if all is conrrect so far, the Rev Primer will be 47bp which is the longest one I have seen, is that available?
Thank you very much!
Linchun
Hi,
BbsI cuts like this (strand cuts marked '!')
5’… GGTTGAGGTCTCT ! AAAAGC GTCTTC CT … 3’
3’… CCAACTCCAGAGATTTT ! CG CAGAAG GA … 5’
to leave a four base 5' overhang.
I am assuming that the way this is presented that you are inserting the gene to the 5' of this sequence, so if you want to re-generate the BbsI site 'as is' your reverse primer will have to be ;
5' AGGAAGACGCTT TTA XXX XXX XXX XXX XXX XXX 3'
the TTA in italics will be a ready made stop codon (TAA in fwd direction) for you and the Xs will be the reverse complement of your last 5-6 codons as appropriate for your primer Tms. Useful tool for reverse complement here:
http://www.bioinformatics.org/sms/rev_comp.html
I also have no idea how close to the ends of the PCR products BbsI likes to cut as I cannot find any data on this enzyme so it may be worth adding another 6 bases to the 5' end.
Is this what you want to do? or would you prefer to add another site so that you can clone the gene out more easily in future? or is the 3'end of your gene to be in frame with a 3' fusion?
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