Digestion or Ligation problems, Help! - Help!!!!!! (Jul/14/2006 )

[font=Arial]Hi, i hope that someone can help me, right now i am a little desperate, i am trying to ligate mi insert that is 2 Kb (Not I/Bgl II) into pShuttle-CMV (Not I/Bgl II), i don't know what am i doing wrong, i gel extract my insert after digestion and also y dephosphatase my vector and after that i clean it with phenol and after i precipitate it, and i ligate for an overnight at 15 C, the competent cells that i am using are JM109 from promega, but i can not have any colony in my plate with the insert neither in my control plate, can anyone give me some tips!!!!!!!!!!!!!


PLEASE, MY P.I. REALLY NEED THAT RECOMBINANT!!!!!!!!!!!!!!!!

HELP!!!!!!!!!!!!!!!!!!![size=3]

Hi,

Don't have to dePO4 the vector since you're using 2 REs. If need be, just gel isolate and purify with kit (e.g. with sillica filter) rather than phenol extraction and ethanol precipitation. Residual phenol will cause problems.

Anyway, I always PCR my product in final vol 50ul. PCR purify with kit. Normally the purified PCR product will have a conc of ~30 ng/ul (vol 30 ul). RE digest with 2 REs using MultiCore buffer (Promega) for 1 to 2 hours (5U/ug DNA or simply 1 ul each RE). Run gel, isolate and purify with kit.

As for vector, 1 ug vector in final RE rxn vol 50 ul. RE used is 5U/ug DNA, or just simply add 1 ul of each to the rxn. Incubate at 37C, 1 to 2 hrs. Run gel, isolate and purify with kit (Note: I prefer gel isolate rather than PCR purify because it ensures undigested vector is eliminated).

Ligation: 150 to 200 ng vector + purified digested PCR product (I would normally add half of my purified product. Too lazy to calculate the 1:3 ratio and it works every time). Ligation is 1 to 2 hrs at RT.

Transform: 3 to 5 ul ligation rxn product and appropriate vol of competent cell. REmember to include positive control for transformation, e.g. supercoiled DNA.

OH YA. If you did all that, just make sure the antibiotic u used is appropriate, e.g. KANAMYCIN instead of AMPICILLIN. Note: pShuttle-CMV is KANAMYCIN resistant.

Cheers

Since you didn't get any colony with your control (only vector, right?), your transformation would be wrong.

Prepare frech competent cells, check transformation conditions (esp. when it's electroporation) with freshly prepared vector, and check the antibiotic concentration. (isn't it too high?)

If there is no problem with transformation, check your vector before ligation by running a gel. In many cases, plasmids are contaminated with nuclease that results in 'digestion into bps' after RE treatment. You'd better check your insert, too.

Hi! I agree on the fact that your problems are in the transformation step... I can tell u my experience with the JM109 cells is not good...actually, I wasted 3 months trying to transform those cells with a pGem vector (no ligation in the midle, just the comercial vector), and the most succesfull I have ever been with those cells came out to be 2 blue colonies. When i switched to Top10 cells with the exact same plasmid preparation, the whole plate was covered with blue bacteria. So, if you can, try changing these cells, maybe you are having the same problem I was.

I have a question: the JM109 cells that u r using the ones that promega sells already in a competent state?

agree - def a transformation issue
not sure how experienced you are, so ignore this if it is below your level:
1. check the temp on your water bath carefully if you are heatshocking
2. the time is critical, so use a timer 45 -50 secs MAX with NO shaking
2. SOC is good for recovery - NO antibiotics at this stage
3. be gentle with the bugs when you pipette them

and as MSGs said - get the antibiotic right - such a simple mistake that i'm sure we;ve all made at some point!

yes, the cells that promega sells are already competent. and are not so good, i cahnge the competent cells for DH5alpha that i made and i could have my recombinant.