random cleavage of plasmids - insertional mutagenesis (May/07/2009 )
My first post, very specific, I hope someone can help...
I would like to insert a specific stretch of 21nt into eGFP at a random position. How to cleave the plasmid randomly? DNAse1 cuts after C or T (not random), isn't it? A transposase integrates DNA rather randomly, but I can not define the inserted nucleotides. Cer(IV) salts sound promising, has anyone some experience?
Any help or suggestions greatly appreciated!
Ok, it is possible that I misunderstood, but….
You are after a random position, so why don’t you just pick unique restriction site in that plasmid/gene, digest it, AP treat it, and ligate that vector with two primers one being your specific 21 nt sequence with unique enzyme overhang at 5’ end and the other primer being your 21 nt sequence with unique enzyme at 3’ overhang.
These two primers have to anneal and produce overhangs, imitating digestion.
Thanks for your answer! There are some unique restriction sites in this plasmid, but those are limited. I was thinking more of a non-restriction enzyme based method, so that I can insert DNA at a random position, ie after any nucleotide of the ~700bp eGFP sequence. So I'd need some unspecific cleavage of DNA. Any suggestions? Thanks again!