Designing primer for Banana mild mosaic virus - (Jan/13/2007 )
i've an unpublish sequence of the coat protein of banana mild mosaic virus. i hv tried some primers but it doesnt give me the sequence that i wan. hope to get some advice in dsigning primers for my sequence.
thanks a lot.
if you have the sequence that you want, you design primers based on that sequence, either by hand or by using a programsuch as primer3.
when you run a PCR with the primers, do you have multiple bands?
is the band you see on the gel the right size?
do you then clone in into a plasmid backbone, and sequence it from that?
if you could give a bit more detail, we'll be able to help you more.
thanks for ur reply.
yes. i got multiple band and i've got my sequence size (bout 700bp) according to the ladder. i did gel extraction but after that no band is seen after the extraction. i've ligate my PCR product with obtain from Gel Extraction (GE) into the vector and transformed it into the competent cell. i grow it at 37 celcius for 16 hours. no growth is observed. i unable to proceed to pick single colony and do the plasmid extraction. wat do u think it is the possibility and how to overcome it? i've try to read the OD and there are something in there. but i dono whether the thing is it that i want.
by the way, are u a researcher? what u specialize in?
ok, if you have multiple bands, that means you're PCR isn't very good. you should try to optimise it so you see only one single band... but any way...
do you have RE site on the ends of the primers which match up to the vector?
is it a blunt or a sticky end ligation?
there are a whole host of issues that arise from ligations.
if you don't have growth, it is either the ligation that has failed, or there could be a problem with the plates, or the bacteria.
and what do you mean you read the OD? is this on the ligation reaction, or something else?