NheI, EcoRI vector double digest - Troubling getting second enzyme to cut (Aug/21/2007 )
Hello. I'm new to cloning, and I'm trying to ligate a 1.8 kb insert into a 5.7 kb vector (modified version of pET15b). I'm trying to digest using EcoRI and NheI enzymes using different cohesive ends. If we forget about the insert, I'm having trouble digesting the vector. Both enzymes are able to cut the vector efficiently, as I can see from my gel. However, I can't seem to get the second enzyme to cut. All enzymes and buffers come from NEB. I've tried double digests as well as sequential single digests using two methods:
1. Cut the vector (from Qiagen MiniPrep from DH5A cells) using NheI in EcoRI buffer with BSA (as recommended by NEB for double digests). Incubate 37C for 2.5 hours. Heat inactivate 65C for 20 minutes. Add EcoRI and incubate again at 37C for 2.5 h. Add CIP for the last half hour, keeping an aliquot as a control without CIP. Use the Qiagen PCR cleanup kit to clean the sample, and then set up ligations. I've gotten no colonies on the transformation with CIP, but lots of colonies without CIP. I use 1 ul of enzyme in 60 ul of reaction, and then add a second ul halfway through. I use 0.5 ul CIP.
2. Cut the vector using EcoRI in EcoRI buffer. Incubate 37C for 2.5 hours as before. Use the Qiagen PCR cleanup kit. Digest with NheI, again for 2.5 h at 37C, this time in NEB buffer 2 with BSA (recommended for digests with NheI alone). Dephosphorylated with CIP as before. Cleanup with PCR cleanup kit. Volumes are the same as above. The results from the transformations are also the same.
Now, the cut sites are only separated by 6 bp (GCTAGC-ATGCAT-GAATTC). NEB says that NheI should be able to cut a vector cleaved by EcoRI with the two cut sites within 1 bp of each other on their web site, however. The way I'm interpretting the transformation results is that each enzyme will cut the vector the first time, but the second digestion isn't working. Any ideas why not?
Firstly I would simply use NEB buffer 2. There is no need to use special EcoRI buffer anymore.
Digest first with NheI, perhaps for 3hrs in either NEB buffer 2 or 4. Then add EcoRI. THere is no need to have an intermediate heat kill step or purification step. Digest for a futher 3hrs.
Then gel purify.
Find out how much DNA you have and dephosphorylate appropriately.
I believe the main cause of failure is not the digest but the dephosphorylation. Excessive dephosphorylation will damage your DNA ends and render said vector unligatable. 30 minutes is far too long (unless you have bucket loads of DNA). How much vector are you dephos?
the rule of the thumb is
1pmol DNA, is dephosphorylated by 0.1U CIP in 60mins at 37 Celsius in a volume of 50ul.
NEB CIP, is about 10U CIP per 1ul. So for most dephosphorylation I actually need make a dilution to work with.
Thanks for your response. I'll try with less CIP. But what I don't understand is if that is the problem and not the digestion, why am I getting colonies on my control plate that has the double digested vector without the CIP treatment? That's why I initially thought the problem was with the second digestion.
how many colonies are you seeing from the control plate? 100s or 10s. If the number is low, one tends to assume the restriction enzymes works. Furthermore, the enzymes that you are using are good enzymes, they behave well. So the eye of suspicion tends to fall on them last.
Typically, I'm getting in the 40-50 range. Roughly the same as the single digest ligation control (cut with one enzyme, then ligate). When I've tried to ligate the insert in, I've also gotten back the original vector that I'm trying to get the insert into.
1. It's difficult to see which one of the enzymes is not working if you only have a 6bp difference, so you need to make sure both your enzymes work - ie digest your vector with each enzyme separatly then run a gel.
2. If both enzymes work, then obviously your double digest isn't working. One possible problem could be that the glycerol concentration of the enzymes is too high in your digest, it should never be more than 10%.
3. Are you sure you have 6 bp between the cutting sites? Don't rely on what the people who gave you the vector say, unless it is a company bought plasmid. You need to have the actual sequence, so do a sequencing.
4. If you are sure that there are 6 bp and both your enzymes work separatly then you need to consider that the enzymes don't work together, irrespective of what NEB says. Then you would have to consider using other cutting sites, or maybe PCR-primer cloning or subcloning a small insert into one of the sites, so you'll have more than 6 bp in between the sites.
Hope that helps
I tried again using a different batch of competant cells, and I'm now getting 100s of colonies on the double digestion plate without CIP dephosphorylation, and similar numbers with the EcoRI-digestion/religation control.
I've checked that both of the enzymes digest the undigested vector individually on a gel. They can both linearize the vector. I don't think I've ever exceeded 5% glycerol concentration. I do have some sequencing results that showed that there are six bp between the restriction sites, consistent with the sequence I was given. And I know that each enzyme only cuts once from the single digests as shown on the gel. NEB says that the enzymes cut within ONE bp of each other, so I'd be surprised if it couldn't manage with six, though at this point I'm not sure what else it could be.