Is dephosphate needed? - (Sep/01/2006 )
Hi, guys, I will digested the vector with two different enzyme. Is dephosphatate needed? Thanks.
I guess I will get this over with. (also refer to http://www.protocol-online.org/forums/inde...showtopic=4947)
Yes, you do need to dephosphorylate. Why? Because
1- restirction digest is never complete. A few molecules will always survive with a single site cut and these can self ligate. If the vector is small <5kb this will happen very frequently. It takes only a few molecules to make a the selection process difficult, and you are already fighting with denatured vector (which don't cut but transform well - a reason to alway gel purify your DNA). So don't make the process any more difficult.
2 - The vector can concatemerise. Two vector molecules can ligate to each other, their ends are compatible afterall.
3- the molecule can self ligate even their ends are incompatable (rare). Strange but true, you can have the rare chew back event which deletes enough bp so that a compatible end forms.
So, save your self the agony, dephosphorylate. (However don't over do it.)
Well, just to raise the question...
I did believe that a vector concatemerisation was a negative issue as 2replication origins were kind of a suicide for a plasmid...
For the third argument... well i've encountered that many times without founding any acceptable explanation.
Hmm... just a thought.
I wonder, do you think it is possible for the vector to religate once it is in the bacteria cell, using the cells repair machinery? And this is the cause for the religation?
I do know something occurs at the junctions where uncompatable ends religate. The area is quite scrambled. (Don't ask why I know... it is a sad story. Somebody made an error in the plasmid design and I was stuck with it. Had to do sequencing work... as I couldn't detect presence of the tiny insertion I had to do)
Concatemeres... I have made and sometimes by accident isolated plasmid dimers based on pBS and pTRC99A backbones. Never seen it with BACs or low copy plasmid types... I guess...it becomes a none issue on big plasmids and low copy number type plasmids.
i dont dephosphorylate. it can interfere with ligation and and effeciency will be lower. i only dephosphorylate if i have single enzyme digestion.
without dephosphorylation step, how was the efficiency of ligation and transformation?--was it sufficiently high enough for screening?
I am also worring about the interference of dephosphorylation to the ligation...
thanks..
VF
I am also worring about the interference of dephosphorylation to the ligation...
thanks..
VF
Oh worry worts (though with a valid point), if the dephosphorylation goes just right, all wows will be naught.
I believe many of the problems go away if lots of DNA was dephosphorylated. I dephosphorylate a minimum of 2 micrograms, often touching 4 microgrames. Yes... I know... you only need about 100pg for a transformation.... but what can I say... I am not such a good ligater, I like to be able to haemorrhage DNA at every step.
