My battle with BamHI - (Mar/23/2010 )
Actually, sequencing is extremely cheap. We use GeneWiz, which only costs about $8 per sample for a ~1000 bp read (though the first and last 100-200 bp aren't very accurate). We have drop off boxes on campus for free overnight shipping. The only other cost is the sequencing primer (though very often a PCR primer you already have will suffice).
Anyhow, it sounds like you're doing it all correctly, so that is extremely strange. Is there anything oddball about your vector? Have you tried digesting some other, perhaps 'more standard' vectors containing BamHI, and do you have the same problems?
It's starting to sound like it's your enzyme -- NEB generally makes good enzymes, and has a great reputation as a supplier of molecular biology reagents, but maybe their BamHI is not so good? I know the addition of BSA is supposed to enhance enzyme performance, but other suppliers (e.g. Invitrogen, the stuff I use) don't use it -- maybe NEB does because the performance of their BamHI needs the boost to perform adequately? Or maybe you just got a bad batch?
I know we're getting out there in terms of theories, but I would try another vendor's BamHI (if you tell them of your difficulties, they'll likely send you a sample for free; even if they won't, it's a pretty cheap enzyme -- Invitrogen charges $40.20 for 2,000 U at catalog price).
While I was looking up the Invitrogen price, I noticed they include this comment (here) about BamHI:
NEB's page on BamHI says the enzyme is not sensitive to methylation (here) -- but maybe it is? What E. coli strain are you propagating your vector in? Is it a dam and/or dcm methylase mutant?
I also noted that NEB says their enzyme is purified from "a
NEB offers E. coli K12 ER2925, which is mutant in both dam (dam13::Tn9) and dcm (dcm-6), for free with an order (see here and here) -- maybe it's be worth a shot to propagate your vector in such a strain to eliminate DNA methylation as a cause?
I think DyDx's idea of trying to digest another vector is a good idea, too...
NEB calls for their Buffer 3 plus BSA... and georgiadave has already stated that he's tried different tubes of enzyme.
It is interesting that NEB doesn't tell you that it can't cleave some methylated DNA...
As for recombinant vs native, it really, really shouldn't matter -- but it's not impossible.
No one has yet suggested that there could be impurities in your DNA prep that inhibit the reaction during your first digestion, and then are removed by the purification, allowing the second digestion to proceed. You did not say what fraction of your initial RE digestion reaction volume was DNA versus pure water. I'm guessing that you have relatively low concentration DNA, and that a large fraction of the reaction is your DNA, and a small amount is water. This would mean that any impurities in your DNA would have a major effect on the reaction. Things like ethanol would do this, for example.
I thought of that too, phage434 -- the effectiveness of the second digestion could be the due to removal of impurities present the first digestion, or it could be that there just wasn't enough enzyme in the first round -- I decided to test the lack of enzyme theory first, since BamHI seems to be the only enzyme georgiadave's having trouble with, which pointed me away from a DNA purity issue. I suppose it's possible that BamHI is more affected by impurities...
I wonder if it's just a bad batch of NEB enzyme, or it was mishandled in some way, reducing its activity...?
georgiadave -- have you tried a fresh tube of NEB's BamHI?
The vector I currently clone with is p416 (ATCC3 87340). I have seen the same results with all other vectors, including pUC18!
I have read a lot about BamHI but never have I visited Invitrogens website to read their data, I guess I figured that since BamHI has been around for a long while all vendors would generally have the same data. Well after looking at the comment about methylation I guess I'm stumped. If the sequence for BamHI is 5'-GGATCC-3' then Dam methylation is possible but Dcm isn't. From NEB's website: (Dam
The idea of the recombinant enzyme being less...i guess active?...than the native is troubling. Especially since that is what we are doing with cloning...that would kinda dis-credit a LOT of work. However, never say never.
My DNA is from a qiagen mini-prep - I don't use buffer HB but I really don't think that is the problem. I elute in their EB buffer. The kit is from last year (2009). I quantify using a nanodrop that hasn't had any reported issues. Their may be a contaminant in the DNA but it doesn't show up in the numbers. Also, my DNA concentrations are generally pretty high (>=300ng/ul) so my digests include 3-7ul of DNA out of 100ul total. I would think that would dilute any salt, EDTA, whatever down far enough to be a non-factor. I don't think I still have EtOH in my preps...I spin for 2 min @ 13k RPM after wash steps to remove all traces.
My enzymes stay in a cold block @ -20degC. There is the possibility of being mishandled since I mentor undergrads who use my reagents. However, I just opened a 'new' tube - it was in the backup RE cold block at the back of the freezer, it wasn't just ordered. It expires 7/10.
OK, I don't think it was DNA contamination, given your high concentration. Dam and dcm and cg methylation do not inhibit BamHI cutting (Rebase). I'm out of ideas.
I would try an enzyme from a different vendor. For instance you can try the Fast digest BamHI from fermentas, upon request they deliver 10µl of every enzyme for free.
georgiadave on Mar 24 2010, 07:08 AM said:
xul DNA (2ug)
10ul Buffer (10x stock)
1ul BSA (100x stock)
xul water
2ul BamHI
Total volume: 100ul
You're cutting in a very high total volume. I usually have a total volume of 20-40µl.
DocFlow on Mar 29 2010, 10:18 AM said:
georgiadave on Mar 24 2010, 07:08 AM said:
xul DNA (2ug)
10ul Buffer (10x stock)
1ul BSA (100x stock)
xul water
2ul BamHI
Total volume: 100ul
You're cutting in a very high total volume. I usually have a total volume of 20-40µl.
True, I only use 20µl with 1µl or max 2µl Restriction enzyme.