question for all you cloning gurus... - (Jan/05/2006 )
So, here is my question:
has anybody ever tried to ligate an AvaI sticky end (C'TCGGG) to an Xho I sticky end (C'TCGAG) when the Ava I site does not have the same R? they are chemically similar and should still be slightly sticky with each other...has anyone ever tried anything similar?
I am assuming the frequency would be pretty damn low, but I am curious if anyone has attempted such a feat
Never tried it, but am very curious as to whether or not it would work.
The other route would be just to blunt end them and ligate that way.
Never tried it. sometime it's already too difficult to ligate correct ends !
But I agree with pBluescript, I would better blunt ends. And I too am curious to know if it works.
I too never tried such thing but in some negative contols where only one nucleotide is different and a random sequence I do get success only different nucleotide. So I assume it should work though at lower frequency.. all the best.
why I asked...I am on a third trip through a tricky cloning. if I don't get my clone by early next week, I will have exhausted all the tricks I know. I have thought that a different vector may help, and that is as close as I can get with the tools at hand...otherwise I'll have to take the time to buy stuff
I'll probably just buy stuff anyways, mostly I was curious if it was possible. the clone is pretty important and I would like to get it faster
Thank you for all your help!
Don't they have PCR cloning vectors based on a Taq overhang of just one A base, where the vector just has an extra U or T for cloning?
I tried one of these once and it seemed to work all right so I can only imagine your "mostly complementary" strategy should work, maybe even a bit better.
Can you PCR your insert using primers that have whatever restriction site your vector needs added to their 5' ends? Does your insert contain internal sites for whichever is your vector site?
Well, I designed the primers to give me BamHI/XhoI overhangs, which I am using to clone into pET14B to make an expression plasmid (in-frame, his-tag, all that good stuff)
supposedly someone else got this to work (I am following the methods section of the paper) to subclone this gene from gDNA; I used the same strategy to clone another gene a few weeks ago and it worked quite easily
the only other vectors I have in my possession right now do not carry XhoI sites (or isoschizomers the closest is an AsaI in one of the vectors, with the 3/4 overhang. I am doing my transformation today and will be screening colonies Tuesday, but if I don't get it this time I will have to change tactics. I think I will probably end up ordering an easier cloning vector, like a TOPO or something like that; I would be concerned about wasting a bunch of time with the 3/4 overhang....although I too think it would probably work. I will try it if it takes too long for my order to come in
OH, hey, I also like pBluescript's suggestion to blunt the ends. that might actually be easier, and I could probably borrow some Klenow from a neighbor and get there faster...blunt cloning can be a pain, but I suspect the problem arises from the proximity of the Bam/Xho sites on the vector and most of my vector is only cut once...a blunt cloning would bypass that problem, I'd just have to sequence more to be sure of the orientation