subcloning - (Feb/02/2001 )

Any suggestions regarding a subcloning problem?

I created 5’ deletion constructs of a fragment of interest by PCR. The template is the entire fragment of interest subcloned into the MCS of pGL2Basic (Promega) between the SacI and BglII sites. Forward primers are complementary to the fragment of interest with a SacI site linked on the 5’ end with a preceding 3 base cap. Reverse primer for all amplicons is complementary to the part of the plasmid downstream to the MCS, and has a BglII site on the 5’end with 3 base cap. The strategy is to digest the amplicons with SacI then BglII (ethanol precipitation in between, as the two restriction enzymes require mutually incompatible buffers), separate on gel, extract, and ligate into a vector prepared by digesting the template in a similar way, finally transforming DH5alpha E Coli cells.

We have changed the gel extraction kit (Quiagen silica gel filter to Genomed Jetsorb), changed TAE brand for gel.

Because of concern that the SacI site was not being cut, as only 3 bp separate it from end of DNA strand, we AT cloned the PCR amplicons into pGEMtT-Easy (Promega), obtained plentiful clones, digested with both enzymes and verified that the inserts were properly cut out by running the gel, and religated into the pGL2Basic derived vector. Still no go.

We have tried a different brand of T4 ligase. Worst of all, the positive control plasmid worked in the initial attempt, suggesting no problem with competent cells, ligase or lab problems (“bad water” or such). It seems the problem is in the DNA.

You run a wonderful service which I have learned much from.

Thank you

Peter

One thing that may be worth checking is whether the PCR product cloned into your pGEM vector is correct. You should therefore sequence the DNA, checking that the restriction site is actually present in there, insteadof just by restriction digest (because the pGEM also has a SacI site I think, therefore the BglII need not necessary be cut). Another thing I often do when doing a double digest is to run parallel reactions with single enzymes only to make sure that the plasmid is completely digested. When doing ligation, I sometimes do a dilution series (5X dilution and then a further dilution) together with a test ligation without any insert (this will find out if your vector is properly digested).

do you havw a problem with the pGL vector prep as this sems to be the point where your problems begin!

HiTry using the protease minus strains for expressing your protein. Good LuckMomin

Someone here gives you good thing to try with negative controls (activity test for enzymes,digested plasmid ligated without insert, etc.). You should try this. I just want to add some information on two points you mentionned:
1- you don't need to precipitate your DNA between the two digestions. Sometimes, residual alcool can severely affect enzyme activity. With SacI and BglII, you can do your first digestion with SacI using a 1X concentration of Y+/tango buffer (Fermentas) or One-Phor-All (Pharmacia, pretty much the same thing). Before doing your second digestion with BglII, just adjust the final concentration of buffer (the same buffer) at 2X. No inactivation needed, that should work fine.
2- SacI is supposed to cut very well the end of PCR products with 3 bases extension. I don't think your problem is there.

Also, did you pay an extra-attention to the ration vector:insert you used for the ligation? It can sometimes have a lot of influence.

Sounds liek the pGL3 is the problem.
How close together are the two sites in the MCS, if they overlap, you may not be able to cut them both. Either this, or one of your enzymes may not be working correctly, check this by digesting with each enzyme independently. What you're trying to do should be relatively simple, is there no way to subclone your sequence from your TA cloning vector into pGL3?