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My battle with BamHI - (Mar/23/2010 )

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pito on Mar 29 2010, 01:00 AM said:

DocFlow on Mar 29 2010, 10:18 AM said:

georgiadave on Mar 24 2010, 07:08 AM said:

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.

-Dukey-

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.

-phage434-

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)

-DyDx-

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.

-Dukey-

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.

-DyDx-

DyDx on Mar 31 2010, 07:08 AM said:

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.

-Dukey-

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.

-perneseblue-

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!

-georgiadave-

@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!

-Questionner-

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

-Bio-Lad-
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