Is my restriction digest not cutting properly? - (Oct/06/2006 )
Ok this is going to be a long post so sit yourself down and prepare to be boggled by this mystery. I am currently trying to insert a 2.2kb insert into pcDNA 3.1(5.4kb). The pcDNA 3.1 I have currently has a 1.6kb insert residing in it. I cut with KpnI and XhoI (all NEB) using NEB buffer 2 overnight at 37 deg C. When I run the digestion reaction on a 1%agarose gel I get 2 bands one at 5.4kb(vector) and one at 1.6kb(original insert). I cut out the band using a new razor blade cutting the gel right on the 5.4kb fragment so the blade only touches the vector. I then run a Qiagen gel extraction kit and run the purified product on a 1% agarose gel. I get a nice clear product only at 5.4kb. I then proceed to do a negative control for my vector, so I ligate my vector by itself. After this is done I do a transformation and get tons of colonies in my supposedly negative (vector only) plate. I have picked some of those colonies and miniprepped them and every colony has the 5.4kb fragment(vector) and the 1.6kb (original insert). This lead me to believe that perhaps my RE were not working properly so I used RE's from another lab and got the same result. I thought maybe some uncut vector may also be in my sample so I transformed the purfied sample without and ligation. I got no colonies, which means that there is at least some cutting happening. My next guess is that I will have to do a SAP digestion and then ligate and transform, so if there is any singlely cut vector+original insert it is dephosphorylated and will not religate to itself. This problem suddenly happened because previously I had no problems like this with cutting my vector. Any ideas would be appreciated because I am going crazy trying to get this to work.
Digest the vector using each enzyme separately for 1-2 hr. Then run on a gel and see which one is working poorly (I guess kpnI because it has only 75% activity in NEB 2). Hopefully you will see linearized vector and some uncut vector for one enzyme. Then just cut out the linearized vector, gel purify, cut with the second enzyme and gel purify again.
Yeah I tried doing a sequential digestion and got the same result. I am kind of at a loss.
Don't be too stressed about this.
It is just a case of single cut plasmid being pulled along with double cut plasmid.
I am going to bet that the KpnI/XhoI cut plasmid was run as a rather concentrated solution.... too much DNA in too small a lane. And on a 1% agarose, there isn't that much difference between 5.4kb and 7kb. So pull down would easly happen.
IF the problem is rather bad, you can do another digestion and gel purification on the purified DNA. As was suggested dephosphorylation would help too. Just don't over do it
Here is my recipe for my Restriction Digest:
4 micrograms of vector (usually 10 microliters)
10 microliters of 10xBSA
10 microliters of Buffer 2 (NEB)
2 microliters of KpnI
2 microliters of XhoI
66 microliters of sterilized water
I then run all 100 microliters on the gel in a large lane. (Actually it is 4 small lanes that I combined into one big lane with tape on my comb, 1 lane generally can take 20 microliters)
Do you think that it is too conc.? Should I reduce the amount digest per lane?
I am currently letting a restriciton digest to go over the weekend and then doing the gel extraction on Monday.
Thank you for your response I appreciate it a lot
I am currently letting a restriciton digest to go over the weekend and then doing the gel extraction on Monday.
It sounds a bit concentrated, (I need to see a picture of the gel to be sure... but is the cut DNA band narrow, and the other DNA bands on the gel well sorted, no smearing, or overly wide?) As for cutting less... well how about sticking more lanes together, 6 or 8 would be fine. I myself like cutting lots of DNA to tie me over any DNA losses.
AS for the over the weekend digestion... I am a bit iffy over that, I tried that once. Started the digest on Friday. DNA was fine on Saturday. But by Sunday my DNA was showing degredation. How long the DNA will last depends on its purifty... but still... based on my experience.. I would not keep the digest going for that long.
This is probably not your problem, but you should note that the BSA is 100x not 10x, and you are using 10x as much as recommended. I definitely agree about the weekend digestion. Digestion is mostly done in 10 minutes. Leaving DNA at 37C in an enzyme friendly environment for long periods does not yield healthy DNA. If you must do it over a weekend, do it in your PCR cycler and chill to 4C after 2 hours.
Also, you want to make sure you are not zapping your DNA with ultraviolet light during the gel isolation. Make sure you are using long wave UV (365 nm) or better, blue light (Clare Darkreader).
If your background problems persist, you can PCR your backbone (putting in whatever restriction sites you need), cut the template DNA with DpnI, mix with your insert, cut, religate, and go. Zero background.
in addition to suggestions by others, i would recommend to run ur gel longer so that u dont have the single cut vector contaminating the double digested vector when cutting it out.
Hi,
I have a very similar problem (only with bgl2, mlu1 and pCAT3 basic). Yesterday I digested singly and double, ran in 1% agarose for a very long time, and the double digest did indeed separate into to distinct bands - one ~500bp (insert length) higher than the other. Bgl2 didn't digest 100% even though I'm using buffer Y+tango 2X.
The ammounts of DNA I used were very small. assuming I use a higher agarose concentration, should I be able to get clean double digested DNA by letting the two bands separate?