Hello, I want to ligate a 50 bp PCR fragment but according to the
manual of the promega kit I am using for DNA isolation there is only
a 2^% DNA recovery for a fragment this small...
As the 50 bo fragment contains a Bah Hi site I thought I might therefore
pursue the following strategy:
1) PCR up a 1 kb fragment of DNA that enconpasses my 50 bp fragment.
2) Isolate this 1 kb fragment using a kit
3) digest this fragment with BAM HI which will give me a 50 bp
fragment bounded by Bam HI sites (as there is one in the 5' primer)
and a 950 bp fragment which has one side cut with Bam HI.
Then I thought I'd run this on a LMP gel and cut out the 50 bp
fragment.
4) To this I will then add i) dephosphorylated Bam HI vector,
ii) ligase.
5) I will then leave this for a while at 14 oC (or whatever)
purify the DNA and tansform some bacteria...
What I want to know is : will this work? Has anyone else done ligations
in LMP agarose? If so is there any considerations of which I should
be aware?
Thanks for any advice or information in advance,
gary morley
