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My battle with BamHI


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34 replies to this topic

#16 georgiadave

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Posted 24 March 2010 - 08:00 PM

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 [G meATC] ) and (Dcm [C meC(A/T)GG] ). I am using XL1-BLUE MR supercompetent cells (here) because they are listed as beneficial when transforming eukaryotic DNA (I am), XL10-GOLD Ultracompetent cells (here) for the really hard to get constructs, XL1-BLUE Supercompetent cells (here) are standard competent cells, and XL1-BLUE competent cells (here) for "routine cloning"...whatever that is! None are mutant for Dam or Dcm because I never thought it an issue. Plus, keeping dam- and dcm- is difficult since they are Cam resistant via a transposon! Anyway, that is an idea to try Homebrew - thanks!

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.

#17 phage434

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Posted 24 March 2010 - 08:17 PM

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.

#18 tea-test

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Posted 25 March 2010 - 12:20 AM

I would try an enzyme from a different vendor. For instance you can try the Fast digest BamHI from fermentas, upon request they deliver 10Ál of every enzyme for free.
tea-test: The artist formerly known as Ned Land

#19 DocFlow

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Posted 29 March 2010 - 12:18 AM

A common setup would be:
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 20-40Ál.

#20 pito

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Posted 29 March 2010 - 01:00 AM

A common setup would be:
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 20-40Ál.


True, I only use 20Ál with 1Ál or max 2Ál Restriction enzyme.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#21 Dukey

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Posted 29 March 2010 - 01:05 PM

A common setup would be:
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 20-40Ál.


True, I only use 20Ál with 1Ál or max 2Ál Restriction enzyme.


Yep the volume is WAY to high for this amount of DNA. I never go above 20 ul for any single digest and try to keep each digest to around 1 ug of DNA. If I have to digest more, I will set up several small reactions and either gel purify each and then pool, or I will pool them and precipitate them as one. Digests in more than 20 ul volume are usually horribly inefficient.

Edited by Dukey, 29 March 2010 - 01:05 PM.


#22 phage434

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Posted 29 March 2010 - 03:37 PM

I have no clue what you could possibly believe the mechanism is for this "fact." Small volumes invite problems with poor control over amounts, high concentrations of impurities in DNA, high amounts of glycerol from added restriction enzymes, loss of reagents to tube sidewall binding, and loss of concentration control due to evaporation. High volumes and dilution are your friend.

#23 DyDx

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Posted 31 March 2010 - 06:08 AM

Jesus, just do a 50 uL digest like NEB suggests. It's not that hard. You people are crazy if you think the volume makes that much of a difference, high or low.

(as a chemical engineer I can tell you with some certainty that the volume DOES matter, but it should be pretty much insignificant from 20-100 uL)

#24 Dukey

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Posted 31 March 2010 - 01:36 PM

Hey you ask for opinions and help, don't shout back when you dont like the answers. Don't forget I can do a BamHI digest every single time without any issues so.................

"as a chemical engineer I can tell you with some certainty that the volume DOES matter, but it should be pretty much insignificant from 20-100 uL"

And you call us crazy? You just contradicted yourself in ONE sentance. Unbelievable.

#25 DyDx

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Posted 01 April 2010 - 08:23 AM

I don't think it's hard to understand, nor is it contradictory: volume matters for reactions, but in this case it should be such a small effect that it doesn't.

#26 Dukey

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Posted 01 April 2010 - 04:31 PM

as a chemical engineer I can tell you with some certainty that the volume DOES matter, but it should be pretty much insignificant from 20-100 uL


How is that NOT contradictory? 20 - 100 ul is a 5-fold difference in volume. Either volume matters or it doesnt, simple as that. The absolute volumes involved are not important but the relative difference is. Is 1L the same as 5L? Come on, let's not get ridiculous here.

#27 perneseblue

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Posted 01 April 2010 - 06:55 PM

here are my two cents worth.

Yes, volume is important. I agree with phage434. The smaller the volume the more prone you are to problems especially those caused by evaporation and pipeting errors. 20ul is small compared to the volume of 1.5ml tube. It take only a small lost of water to change salt concentration drastically in a 20ul volume.

Big volumes are your friends. Though your boss, who pays for those enzymes you are using, will probably disagree. So I use big volumes for important things like cloning. And small volumes for unimportant diagnosis restriction digest (10ul).

For cloning purposes, cutting lots of DNA is, in my opinion, a good thing. The more DNA, the better. At every step in the cloning process, you are liable to lose DNA. If you start with a small amount of DNA, a bad day or a mistake, and you may lose enough DNA to make your day even worse. Below a certain concentration, DNA will not precipitate in ethanol. Butanol dehydration may not be possible. It also leaves you open to the common problem of "After ligation, my DNA went missing. Or I have no colonies".

I cut around 5ug of DNA in 100ul. Yeah it is a waste but it is better to have too much material then not enough.

As for BamHI from NEB, I wonder if anybody has had the feeling that NEB BamHI works better with more then the standard amount of BSA. I didn't really test it, but when I increased the BSA concentration from 1x to 5x, I felt that the NEB BamHI cut more reproducibly.

Personally, I now cut BamHI in 5x BSA, especially for big cloning restriction digest.
May your PCR products be long, your protocols short and your boss on holiday

#28 georgiadave

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Posted 02 April 2010 - 10:17 AM

If I had 5ug DNA for every cloning digestion that would be ideal - 2ug doesn't require as many mini-preps :)

I have not thought of adding extra BSA, that is definitely something I will play with. Thanks!

#29 Questionner

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Posted 14 July 2014 - 06:44 AM

@georgiadave

Just wanted to say thanks for you summary post and all the follow up comments from 2010. I too have been battling with BamHI and resorting to double digestion to reduce self ligation/lack of cutting. Nice to see I'm not alone!



#30 Bio-Lad

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Posted 18 July 2014 - 05:08 AM

I use the BamHI-HF version from NEB and have always had great results.  I digest anywhere from 1 to 7.5ug in a 40uL reaction (using 1uL BamHI-HF and CutSmart buffer and no extra BSA) and have never had any problems with it.  I haven't used their non-HF version in a very long time, though. cool.png


Edited by Bio-Lad, 18 July 2014 - 05:12 AM.

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