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

i'm not sure how to calculate DNA concentration, my A260 reading average is 0.160, I use 20ul of DNA with 180ul of sterile water for the spectro, and i have a total volume of 200ul. Usually I take my DNA concentration reading off the spectro, but i dun think that is accurate

I would not increase the digestion time to overnight, as you may see star activity.

I agree with all suggestions put forth by Homebrew, though!

NEB ships a custom buffer with BamHI. Buffer 2 has 100% activity, but I think it has worse star activity. See http://www.neb.com/nebecomm/products/productR0136.asp

You're right, phage434.

I just looked at their Activity in NEBuffers chart for Bam HI, which shows 100% in NEB buffer 2 (I use Invitrogen Bam HI, which doesn't use a special buffer).

The buffer that ships with NEB's Bam HI has three times as much NaCl compared to NEB's buffer 2, but is otherwise identical:

1X NEBuffer BamHI
10 mM Tris-HCl
150 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C

1X NEBuffer 2
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C

what is the ug of your DNA?

Not good, there isn't any digestion, and it seems that the DNA samples are smearing on the gel. I incubated for 2 hours, and I used the NEBuffer BamHI provided along with the Bam HI enzyme. Incubation was at 37 degrees. Followed homebrew's digestion volume. My gel was 1.75% and I run it at 120V for 45mins. I uploaded the picture, see what u can make out of it

yeah, I would expect to see less DNA in the wells and more DNA smearing down the lanes

are you certain you don't have to worry about any solvents inhibiting the reaction, from that kit?

My marker size is 100bp, showing 11 bands ranging from 1500bp to 100bp, and i'm pretty sure that the samples and inhibitor free, i did not use any phenol, choloform, and I made sure to follow the additional step to complete remove ethanol. Beside, my RAPD-PCR worked, i showed some bands but not too clear. I believe it might be due to the purity of the DNA, though the DNA samples has an average reading of 1.7 (A260/A280).

So, you need a gel which shows your uncut DNA, your DNA cut with BamHI, your DNA cut with EcoRI, your DNA + some plasmid (pUC19?) uncut, your DNA + same plasmid BamHI cut, your DNA + same DNA EcoRI cut, marker lanes on both sides.

For EcoRI I really mean any other common enzyme which you have sitting around (in its correct buffer). If you have enough lanes, then showing cutting of the plasmid alone would be a bonus.