My single restriction enzyme digestion doesn't work - Digestion problem, no cutting. (Sep/18/2006 )

Hi, thx in advance for helping.

My problem is that when I perform single restriction enzyme digestion, enzymes are from NEBiolabs, incubate for 2 hours and run on 2% gel, i see no digestion/cutting at all. I only see my genomic DNA. Right now, I'm using Qiagen DNeasy tissue kit for my DNA isolation, so i'm not too worried about residual salts and phenol:chloroform. The enzyme I use is BamH1, NEBuffer 2 and BSA all provided by the NEBiolabs. Really troubled over this, Thanks for your help.

FYI, this is the components and the volume i use for my restriction digestion.

DNA sample - 15ul
BamH1 - 5ul
10% BSA - 5ul
1x NEBuffer 2 - 25ul

Total volume is 50ul

I really dun know whats wrong with the reaction, pls help

If wld be good, if u guys can check on my components list, see whether my volume for any of the components is wrong.

Hi,

the only thing I see is that you use 25µl of 1x buffer. In a total volume of 50µl it is 0.5x then...
Maybe this is one point? In the kit there is usually a 10x buffer - so you need 5µl of this in your reaction.

Or maybe 2 hours is too less time, just try another time over night (37°C). Maybe it will work better then.

Good luck!

Chakchel

My first thought was that, maybe there is something wrong with your enzyme.
So if you know someone who has some functioning enzyme (which they have tested successfully) use that.
And I do my restriction as follows:
Minimum of 100 ng DNA , let's say 1µl DNA + 1µl Buffer + 1µl BSA (the one provided by NEB) + 1µl enzyme + 6µl water.
So according to my protocol you use too much buffer. And what about the concentration of your DNA? You say 15µl. How much DNA is that?

I don't know how concentrated your DNA sample is, but 15 µl seems like a lot. Additionally, you're right at the maximum for enzyme -- 5 µl of Bam HI in a 50 µl volume brings the glycerol concentration to 5% final (the enzyme is stored in 50% glycerol), which is right at the maximum -- greater than 5% glycerol is inhibitory. Finally, 25 µl of 1X Buffer 2 in a 50 µl volume means your buffer is at 0.5X final.

I would try the following:

10 µl DNA
4 µl Bam HI
5 µl 10X Buffer 2
5 µl 10X BSA (I assume you've diluted this from the 100X it ships as)
26 µl sterile distilled water

How long are you letting your digestion incubate for?

I think your DNA sample has 5-Me cytosine methylation at GATC sites. Where does it come from? I've had exactly this experience with BamHI. Does your sample cut with other enzymes? Does it cut, for example, with MboI? Or any other enzyme, for that matter.

There is also the possibility that your DNA sample contains an inhibitor, such as phenol. You could test this by adding a small amount of some DNA which you know cuts into the reaction and verifying that the DNA added still cuts in the presence of your DNA sample. I believe BamHI has a custom buffer, which I would recommend you use instead of buffer 2 suggested by Homebrew, but other than that, I would second his recommendations.

Thanks all for the quick reply, HomeBrew I will try that volumes tomorrow when i get back to the lab. I'm isolating goldfish DNA, and i'm going to incubate for 2 hours at 37 degrees. Besides the BamHI, i have other enzymes such as HaeIII, EcoRI, Hinf II. Rite now i do not have any DNA samples with known recognition sites that i can do a control with. Thx for the high glycerol info, and all of your input. I'll try Homebrew's protocol and see whether i can upload my gel run. Btw, i'm not too sure what agarose concentration and voltage i shld run my gel on. Any suggestions ?