HELP! Can't double digest pcDNA3.1 vector - (Oct/07/2009 )

Hello,

Have been trying for a while now to clone a PCR product into pcDNA3.1 vector.

Am using BamHI and NotI.

Using each enzyme separately I see a cut product when run in a gel along side uncut vector (2.5hr digestions).
However in doing a double digest I only get one cut.
Have tried sequential digest too, using Not I, purifying, then using BamHI. Seems that BamHI no longer wants to cut the linearized vector.

NEB double digest tool says that these enzymes are compatible in NEB3.

Any explanations? Or am I just doing something horribly wrong?

Any advice welcome!!

These cut sites are only 50 bp apart, on a 5400 bp plasmid. How are you determining if both sites are being cut?

phage434 on Oct 7 2009, 03:19 PM said:

Testing for cutting:

I can test the first cut, ex. NotI, but digesting it and then running it along side undigested vector.

I then do the second digest, BamHI. This, I can't test on a gel, as you are correct in highlighting that the intervening fragment is only 50bp or so.

So I do three ligations.

1. My "double digested" vector only
2. My "double digested" vector and ligase
3. My "double digested" vector and "double digested" insert

When I transform bacteria with these, and plate onto Amp plates, I see colonies for conditions 2 and 3. Colony presence for condition 2 indicates that there is one cut, that is able to recircularize in the presence of ligase. I see no colonies growing for condition 1, indicating that the vector is liniarized.

Any thoughts as to what may be happening?

Cutting will not be 100% effective, and you will have some recircularization of vector in condition 2. But there may well be good transformants in condition 3. Have you checked to see if some of those colonies are good?

How are you purifying the PCR product prior to RE digestion?

phage434 on Oct 7 2009, 06:02 PM said:

Hi again,

I did a mini prep on condition 3 (when I did the double digest in one step) and did not see my desired insert in any of my picked colonies (I picked 10). I had about the same amount of colonies on bacterial plates from condition 2 and 3. When I did the sequential digestion I also so similar colony numbers and have not yet done the miniprep, not expecting to find anything (also having to wait for a new miniprep kit to arrive in the lab).

I don't purify the PCR prior to RE digestion, but post, using the QIAquick PCR purification kit. I feel as though it's the digestion of my vector that I am having trouble with. (Am not sure how my PCR product is being digested, any way you know of of testing this? The RE sites would leave only about 5bp on either end post digestion).

-Ryang

You MUST purify your PCR product before digestion. If you do not, then the PCR enzymes, buffer, dNTPs are all present to happily extend the 3' end of the cut site, destroying its ability to ligate. This is your problem.

Thanks for the suggestion. Hopefully I'll be successful in the next round!