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My battle with BamHI


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34 replies to this topic

#1 georgiadave

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Posted 23 March 2010 - 01:56 PM

Over the last several months I have been attempting to create a plasmid library for several clones I work with, and I do mean attempt. After reading the NEB manual/catalog I thought that I would be able to quickly troubleshoot any problems and produce my clones quickly, man was I wrong. After the usual problems came and went I continued to have trouble finding clones within the myriad of oddball constructs.

To make a long story short I will cut to the ending.

All of my cloning troubles stemmed from BamHI. Basically I was only partially cutting my DNA, both insert and vector. BamHI looks perfect on paper: fast cutting, efficient ligation record, established in cloning, etc... Not quite. BamHI has the ability to slide along DNA once bound until it finds it's recognition sequence. This is controlled by the salt concentration for the reaction, if the conc is too high then no sliding will occur resulting in a lowered efficiency. This doesn't sound like a big deal since the enzyme is sold with an accompanying buffer, but BamHI has one more trick up it's sleeve: ES complexes that do not result in E + P or E + S. The Km value for BamHI seems to plummet in reactions that have low water and/or high salt; and no, I have no data to back this up. To fix my problem I did something that I didn't foresee having to do, be redundant. For all ligations that use BamHI I digest the DNA, purify, digest a second time with BamHI, gel extract. I also make sure that there is an excess of water, typically I use 100ul total volume. I'm not sure why but this reduced my negative control transformation (digested vector w/o ligase) numbers from 300+ to zero. I was worried about trashing my DNA but adding 0.5ul T4 ligase (with buffer of course) gives transformation numbers similar to the positive control.

I write this because this forum helped me figure out my problem. Hopefully this post will help another who is as frustrated as I was.

After everything I am reminded of something a professor once told me: "don't believe everything you read and question everything."

#2 Ewan McGregor

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Posted 23 March 2010 - 02:20 PM

Thanks, I'll try to do your way next time I clone as I usually have very low number of positive colonies

#3 DyDx

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Posted 23 March 2010 - 04:00 PM

Sorry if there is something I am missing here (I am new, and did not see the posts on this forum where you got help with this), but, honestly, based on the information in this particular post, I think you must be doing something wrong.

BamHI is the RE I use most frequently and I have never, ever encountered problems with it. I get complete digestion in an hour or less at 37C (based on agarose gel visualization). Remember that digesting for too long can cause star activity, though this isn't usually a problem with BamHI. I don't get the greatest ligation efficiencies (which is probably my fault), but I also do not get many (or any) negative control plate colonies, nor do I get false positives on my ligation plates.

Are your DNA samples PURE (i.e. either mini-prepped DNA or purified PCR products)? What are the A260/A230 and A260/A230's?

I think you just need additional purification steps. Everything you said about salts seems irrelevant to me, because the only salts that should be present in your digest are the salts that are in the RE buffer, as well as any salts in your DNA purification elution buffer (i.e. Tris*Cl found in Qiagen's Buffer EB).

Another option is to try out NEB's BamHI-HF (high fidelity), which is now the same price as regular BamHI (at least for the larger volume they sell), which may provide better results for you.

#4 georgiadave

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Posted 23 March 2010 - 04:48 PM

I added responses in blue.

Sorry if there is something I am missing here (I am new, and did not see the posts on this forum where you got help with this), but, honestly, based on the information in this particular post, I think you must be doing something wrong.

BamHI is the RE I use most frequently and I have never, ever encountered problems with it. I get complete digestion in an hour or less at 37C (based on agarose gel visualization). Remember that digesting for too long can cause star activity, though this isn't usually a problem with BamHI. I don't get the greatest ligation efficiencies (which is probably my fault), but I also do not get many (or any) negative control plate colonies, nor do I get false positives on my ligation plates.

My digestions started out at an hour but went up to 3 hours in attempts to allow complete digestion; they are now back to an hour. Star activity has never been a problem. I did have lots of false positives as well as lots of negative control colonies, now I don't. It's wonderful that you get good results with "normal" digestions, I don't, but only when using BamHI.

Are your DNA samples PURE (i.e. either mini-prepped DNA or purified PCR products)? What are the A260/A230 and A260/A230's?

My samples have always been very clean. Typical 260/280 is 1.8<x<1.95 and 260/230 is always >1.8

I think you just need additional purification steps. Everything you said about salts seems irrelevant to me, because the only salts that should be present in your digest are the salts that are in the RE buffer, as well as any salts in your DNA purification elution buffer (i.e. Tris*Cl found in Qiagen's Buffer EB).

Right you are. What I was alluding to was the need for water, nucleases are hydrolases. Also, 37degC causes some evaporation. With both the salt concentration rises. The smaller the total volume the bigger the shift in salt conc upon evaporation of water. I make this point because I know many people who digest in the smallest possible volume, myself was included. Why digest in >=50ul when doing so will require either purification before loading on a gel or taping together wells. Neither are difficult but I avoid both when possible.

Additional purification steps are not needed if DNA is clean, unless our nanodrop is defective my samples were clean after purification.


Another option is to try out NEB's BamHI-HF (high fidelity), which is now the same price as regular BamHI (at least for the larger volume they sell), which may provide better results for you.


I didn't assume that this was going to rock the foundation of modern cloning, as you point out BamHI has been used for a long time. I do hope that it aids someone in their cloning journey. Mainly it's food for thought.

#5 DyDx

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Posted 23 March 2010 - 05:01 PM

Yea, sorry, I just can't fathom why you would be having problems with BamHI, it should just work.

One thing I do is that I ALWAYS dilute my DNA sample to approximately 50 ng/uL (before adding to digest) and make up a 50 uL digest. Every time. This concentration seems to be fully digested quite well, and it is still high enough to be visualized on a gel so you do not need additional purification (unless you need to remove unwanted large fragments, of course).

The reason I chose to do this is that when I was learning the methods, the vector I was using mini-prepped to about 50 ng/uL and I always had good results digesting it, so I have carried this along and it has been successful. I don't know if this helps, but if you are digesting a high concentration of DNA it is something to consider. You really should not have to digest twice to have a successful ligation.

#6 HomeBrew

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Posted 23 March 2010 - 08:05 PM

I do not doubt your observations, georgiadave, and I'm glad you found a method that works for you. I also appreciate you posting your observations for others that might be having similar problems -- that's what the BioForum is all about!

I am a bit puzzled, though. Like DyDx, we use BamHI for cloning in my lab far more often than any other restriction enzyme, and have had very little trouble with it. We use BamHI from Invitrogen, routinely digest for just an hour in 30 - 50 ul volumes at 37C, and always gel purify our digested vector and insert segments through an agarose gel with guanidine HCL added as UV protectant before ligating, but these don't seem to be related to the problems you're having...

How much enzyme are you using? What is your typical digestion reaction mixture composed of?

I understand that you've done digestions with other enzymes without issue, which makes this a very curious problem...

Have you tried BamHI from a different vendor?

#7 georgiadave

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Posted 23 March 2010 - 09:08 PM

Ha, I know. When I finally found this method I was very surprised and upset at the same time - it took a while. The concentration of my stock DNA is higher than 50ng/ul but I don't digest a set volume, I usually digest 1-2ug at a time.

A common setup would be:
xul DNA (2ug)
10ul Buffer (10x stock)
1ul BSA (100x stock)
xul water
2ul BamHI
Total volume: 100ul

All of my RE's are from NEB. This is something that has happened with every tube I have used. I have tried using less enzyme per reaction to get the same results. I have never used enzymes from a different vendor, but this problem only occurs when using BamHI. All other ligations are by the book (ex: KpnI, SalI, HindIII, etc...)

#8 HomeBrew

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Posted 24 March 2010 - 04:09 AM

Have you tried using more enzyme? I usually use 2 ul of enzyme in a 30 ul reaction volume, the same amount you're using in a 100 ul volume.

Your main problem -- negative control transformation (digested vector w/o ligase) producing 300+ colonies -- seems to stem from incomplete digestion, which is improved by a second round of digestion. I suspect you're not using enough enzyme in your initial digestion, and if you upped the amount in the initial round (as long as the amount of enzyme is < 10% of the final volume), the situation would improve.

#9 DyDx

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Posted 24 March 2010 - 07:59 AM

Here is my standard digest, I wonder if you'd see a successful digestion in one step with this formula:

39 uL DNA @ ~50 ng/uL
5 uL NEBuffer
5 uL H2O or 10x BSA (I forget if regular BamHI uses BSA...)
1 uL BamHI
------
50 uL

37C for 1 hour. I believe NEB calls for a 50 uL reaction and I would suggest always using this volume if possible. It's not intuitive, but the volume can have an effect beyond the salts problem you were talking about.


Another possibility, though unlikely, is that your vector is self-ligating even though you are using sticky ends. I have noticed that if I take the time to treat with CIAP, I get 0 control plate colonies, whereas I might get a handful if I don't. I know -- it doesn't make sense, sticky ends shouldn't ligate -- but the cells manage to do it at a low rate anyhow. This doesn't sound like the cause of your extremely high background, but it might help too.

Edited by DyDx, 24 March 2010 - 08:00 AM.


#10 georgiadave

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Posted 24 March 2010 - 08:43 AM

@Homebrew: I have tried digesting with up to 5ul enzyme in 50ul with 1ug DNA to get the same results.

@DyDx: your setup adds 39ul of 50ng/ul DNA - this equals ~2ug DNA...the same amount that I routinely digest. The catalog does call for 50ul digests of 1ug DNA. I generally increase my total volume as my total DNA concentration increases; so for 1ug I use 50ul, 2ug I use 100ul, etc... I do this based on a post by pcrman - I liked the logic behind his theory.

Also, say I do a sequential digest with SalI followed by BamHI with only purifications, no gel extraction. I will get moderate levels (still too high!) of background colonies. Now, if I flip the sequence of enzymes with BamHI first I will get the same extremely high background.

I have done controls by only using SalI once w/o gel extraction to get only 2 negative control colonies but great ligation transformant numbers.

For all trials I grow individual colonies for a mini-prep to check the plasmid by RE digestion; I used BamHI, as well as other enzymes known to cut the plasmid, to make sure the recognition sequence wasn't trashed. This is to check the size of the resulting clone, basically to check for oddball stuff that can happen - all have proper size. The only ones that don't are the partially digested mixtures of vector and insert that I attempt to ligate. After the double digest of BamHI protocol I do not get the weirdo constructs that I do not want. I haven't done sequencing b/c it's expensive :P

Thanks for the replies, this is great!

#11 DyDx

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Posted 24 March 2010 - 10:03 AM

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?

#12 HomeBrew

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Posted 24 March 2010 - 01:16 PM

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:

Does not cleave DNA when the 5 C residue is 5-methylcytosine, but cleaves DNA when either the 3 A or C residue is N 6 -methyl-adenine or 5-methylcytosine, respectively.


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 [sic] E. coli strain that carries the BamHI gene from Bacillus amyloliquefaciens H (ATCC 49763)", whereas Invitrogen's BamHI is "purified from Bacillus amyloliquefaciens H", presumably directly. So, NEB's is a recombinant enzyme, while Invitrogen's is "native". Could this be a contributing factor?

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...

#13 DyDx

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Posted 24 March 2010 - 04:53 PM

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.

#14 phage434

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Posted 24 March 2010 - 05:18 PM

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.

#15 HomeBrew

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Posted 24 March 2010 - 06:47 PM

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?




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