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strange digested vector isolation - (Sep/21/2006 )

Hi everybody. I digested an average size vector (+-7200bp) with BstBI (there´s only one site in the sequence for sure). The problem is, after dephosphorylation and precipitation (0.3M NaAc-EtOH) as usual, the digested vector seems not to be able to enter the agarose gel for band isolation, most of it remains stuck in the well. Any ideas? Should I phenol-chloroform extract?

Thank you very much

-miguelon-

QUOTE (miguelon @ Sep 21 2006, 01:21 PM)
Hi everybody. I digested an average size vector (+-7200bp) with BstBI (there´s only one site in the sequence for sure). The problem is, after dephosphorylation and precipitation (0.3M NaAc-EtOH) as usual, the digested vector seems not to be able to enter the agarose gel for band isolation, most of it remains stuck in the well. Any ideas? Should I phenol-chloroform extract?

Thank you very much



just a very stupid thought,

have u rechecked the gel and the buffer. also the if the gel chamber is functioning.

-scolix-

Hmm... is your DNA soluble? Hold the vial up to a bright source of light, do you see any particals floating in it. Do you use heat to dry your DNA, or leave it drying on bench for a some time?

No?

Well if none of the above, I would agree with you. And try a phenol-chlorofrom extraction. It could well be very heavily contaminated from the digest and dephos step. In which case the above steps may not have worked all that well.

EDIT: Oh, that would just be classic. Reminds me of one incident with a 3rd year student... asked why the miniautoclave wasn't working... it wasn't working because it wasn't plugged in!

-perneseblue-

another quick fix that is often overlooked...use fresh blue juice

there are 3 main reasons, IMHO, for plasmid DNA to sit in the wells:
1. a bunch of contamination in the prep, proteins etc
2. mechanical problem with the gel apparatus
3. something related to pH, often involving the blue juice or whatever your DNA is suspended in

-aimikins-

QUOTE (miguelon @ Sep 21 2006, 09:21 PM)
Hi everybody. I digested an average size vector (+-7200bp) with BstBI (there´s only one site in the sequence for sure). The problem is, after dephosphorylation and precipitation (0.3M NaAc-EtOH) as usual, the digested vector seems not to be able to enter the agarose gel for band isolation, most of it remains stuck in the well. Any ideas? Should I phenol-chloroform extract?

Thank you very much


This is caused by over-drying the DNA so it doesn't dissolve completely when re-suspended.

This once happened to me and at that time I had nothing to lose, so I used gel purification kit to recover the DNA stucked in the wells and runned it again in agarose gel.

Don't dry your DNA for too long and make sure it completely dissolved before you run a gel.

-chick gene-

Hi,

I think it's a solubility problem. Could be bcoz of some contamination in the DNA.

-exploresci-

Hi all. I thought about maybe some problem with this particular enzyme, ´cause I´m using at the same time same buffers, gel chamber, combs...even MilliQ water for other cloning works. And I ran (for twice and just besides the "strange" digestion product) an undigested sample of the vector, that enters nicely in the gel. Cutting the same vector with BamHI or HindIII (two other single-target enzymes in my vector) and equal dephosphorylation and precipitation do not appear to have any problem. So it seems to have something to do with this particular enzyme (BstBI, NEB). Has anybody have any problem like this with any enzyme?

I would also appreciate any suggestion or tips for good plasmid DNA phenol extractions.

Thanks everybody in advance

-miguelon-

EcoKI methylase can inhibit this enzyme. See http://rebase.neb.com/cgi-bin/damlist?eBstBI

-phage434-