problems in a double digestion with NheI and EcoRI - Cloning problem (Apr/06/2006 )

Hello!

I'm having a bit of an issue with two restriction enzymes (brand NEB) in the last step of a cloning, and was hoping someone might have a suggestion. I'm trying to digest my insert and vector with NheI and EcoRI, two unique sites according to the sequences I have.

When I attempted to do a single/double digestion of the two plasmids in the EcoRI buffer (as recommended), I got a single band for the EcoRI only digestion (expected); however, I got an extra band when I used NheI in the EcoRI buffer, and in the double digestion of Nhe and Eco. The single digestion gave me a band of approximately 12kbp (insert) and 9.5kbp (vector), which I can't explain as the respective plasmid sizes are 6.1kbp (insert plasmid) and 4.9 (vector plasmid). In the double digestion, this extra band is the size I would expect if I performed a single digestion of the plasmid, but it looks like a real, clear band, rather than undigested material -- perhaps this interpretation is incorrect?

I then tried the digestion in the NheI recommended buffer (Buffer2 + BSA). I got single bands for both single digestions, but double digestion again resulted in an extra band both for the insert and the vector.

I have not used these enzymes together before, but I have used each in previous stages of the cloning with no problems. I have cut the vector with NheI (alone in buffer 1 and with AgeI in buffer I) with no problems. In addition, these cut sites are not close together -- they are separated by approximately 700bp in the vector and by 2200 bp in the insert. Hopefully someone has an idea of something I could try -- could the extra bands be the result of star activity? Could the enzymes be interfering with one another? NEB doesn't have sequential digestion as recommended, but maybe I should try that?

Any suggestions are appreciated, as a dent is forming from where I've been banging my head into the wall...

most puzzling...could you post a photo?

I think star activity typically shows up as smears and not discrete bands, although that's a possibility; have you tried anything to pinpoint this as a source of the problem?

Are you QUITE certain your DNA is what you think it is?

I believe I have the correct DNA. I did a test digestion of the plasmid that is my insert after the second step of cloning and got the bands I expected. In addition, I used Nhe and Eco separately in the two other steps (it's a three step cloning) with no problems -- they should be the single sites in the construct.

For the plasmid that is the vector, Nhe should definitely be working -- I used it approx 2 months ago to make a single cut in buffer 1 and got a nice band:

The vector cut with Nhe only in buffer 1 is in lane 4.

Here was my first try.

In the top, lanes 2-5 are the insert plasmids, and lane 6 is the vector, and this is my double digestion. In lane six, I though have two bands, 758bp and 4721bp (the one I want). I think the 4721bp is squished in under that larger band. In lanes 2-5 I should have 2280 (the one I want) and 3972.
In the bottom, same order, lanes 2-6 are the Nhe only with extra band(s?), and lanes 7-11 are Eco only, which look like they're supposed to.
This is all in Eco buffer.

Then I compared it to undigested plasmid, in EcoRI buffer

Lanes 2-3 are undigested, insert and vector respectively
Lanes 4-5 are single digest with NheI, insert and vector respectively
Lanes 6-7 are the double digestion -- the band I want in lane 6 is very, very faint, and gone is my 700bp band in Lane 7.


So I tried it in a different buffer.

In Buffer2+BSA

Lanes 2-3 are single digest with Nhe, insert and vector respectively -- they are where they should be
Lanes 4-5 are single digest with EcoRI, insert and vector respectively -- ditto.
Lanes 6-7 are double digest with Nhe and Eco, insert and vector respectively. In lane 6, the highest molecular weight band shouldn't be there. In lane 7, I believe the band I want is actually smushed into the bigger band -- it's only a 700bp difference so they're not all that far apart, and it looks like two bands to me.

I hope what I'm saying makes sense. This whole process is my first cloning... it's been a little frustrating. It looks like the plasmids aren't completely digested, which is why I'm getting the bigger band -- I just don't know why, and don't know how to fix it.

what you are saying is clear (I think!?)

if you look at the 3rd pic, I suspect your problem is what you think it is..you are not getting complete digestion, and therefore will struggle to get the proper bands.

also, I agree with you on a point you made regarding the 4th pic...that larger band in lane 7 looks like a doublet

I would say it's pretty clear that EcoRI buffer isn't doing you any favors on the double-digest (lanes 2&3 look identical to 4&5 on pic 3, which suggests you're not seeing much cutting at all with that buffer)

what are your digestion reaction conditions, in detail please?

I'm making a 20uL digestion with 2uL DNA, 2uL buffer (10x), .5uL Nhe (at 10units/ml), .25uL EcoRI (at 20units/ml) and the rest of the volume made up in sterile water. Because I've been doing multiple tubes for each digestion condition, I haven't actually been pipetting .25ul -- for example in the third image, I measured 2.5x the amount of each ingredient and then pipetted 18uL of the mixture into the DNA.

After I combine everything, I mix it and briefly centrifuge, and then incubate at 37C for 1 hour, which has been working pretty well for me to this point -- maybe it should go longer? I think my biggest issue right now is the vector plasmid -- I might have enough DNA do to a preparative gel and gel extraction of the band I want in the double digestion of the insert, even with the undigested band. I don't think I could cut the band I want out of that doublet though without taking some of the other one.

i'm in a trouble... you've a vial of enzymes as such low concentration??? normally it's 10 000u/ml for Nhe (if stock). Hence, how did you diluted it? and how do you store your diliution?

*moreover you wrote : a 20 µL dilution... But you add 25 µl of EcoRI... So at basis, there is a problem...

No, I didn't say that. I added...

.25uL EcoRI
.5uL NheI

The concentration of the enzymes shouldn't exceed 5%, according to NEB, so I can't have more than 1uL of enzyme in a 20uL digestion. This should be the equivalent of 5000 units of enzyme per reaction.

I did make a mistake in the concentration. I shorthanded to 10,000 u/mL and 20,000 u/mL as 10 and 20. Sorry about that. the conc of the enzymes is:

Nhe 10000u/mL
Eco 20000u/mL

sorry, i didn't see the " . " in the numbers... i though to the fact you didn't add BSA. Searching in the neb catalog, i found the mention "add BSA to the 1X concentration in order to obtain 100%"
Moreover it's also mentionned sthg like BSA does not inhibit restriction enzyme activity.
So i would use BSA.

I do digestions in general cases at 2h. Some slight alterations in DNA purity may affect the enzyme activity. So maybe there is a critical point here.

well, obviously you don't want to digest o/n or you might run into star activity

however, I would increase the time...unless I am in a very big hurry (in which case I add more ~3x enzyme and incubate for 45-60'), I typically incubate 3-4 hrs at 37 without any problems with star activity, particularly when I am being stingy with enzyme and I want to ensure complete digestion for cloning.

there are those who say you never have to cut so long, but I find that I get better results this way overall

and don't forget your BSA (needed for NheI, right?)

hey Fred we cross-posted

great minds think alike