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Cloning Problem - Ligation or Transformation - (May/14/2012 )

I am trying to clone a 1.3Kbp gene into pET21a+. For my experiment, I amplify the gene using PCR and gel digest using BamHI and XhoI and gel purify both the vector (add CIP after digestion) and gene. I performed ligation using NEB T4 DNA ligase at 16degC, overnight incubation. Following ligation, I do a PCR using my ligation (2ul to 20ul reaction) T7 forward and reverse primers and I get 2 bands, band size which correlate to vector with my gene-of-interest and empty vector. However, when I transform my cells with the ligation products, I get about 7-10 colonies. When I do colony PCR on all the colonies, I only get band size for empty vector. So, my questions is that can I say that my ligation reaction is successful and the problem is transformation? Or is ligation also a possible problem? Any suggestion how to get over it? I have been trying for long time, playing with the ratio of the insert with the vector, which everyone in my lab suggest. Thanks.

-shuqinT-

did you set control ligation (no insert, only vector)? If no colonies in control plate then you can confirm that no self ligation (only vector ligation), this happens only because of restriction enzyme. Both are different enzyme, after digesting the vector what was the release size, could you see the release? And more over, no need to do pcr before transformation. After transformation, pick colonies and patch, then do pcr

-christy-

Are you pcr'ing the bam and xho sites onto the amplicon by incorporating the sites onto your primers? That method always sounds like the perfect idea, but having an appropriate amount of overhang after the cut site is not super easy. I've always had to topo-clone the amplicon with the restriction sites into a plasmid first, and then cut from the plasmid.

Also, if you are doing directional cloning using two different enzymes and gel purifying, you don't need CIP on the vector, it simply decreases yield.

I agree with christy, no need to pcr confirm your ligation prior to transformation, do colony screen pcr afterwards. I can totally imagine pcr'ing ligation reactions always giving you positive results even if it isnt :P

Finally, what kind of competent cells are you using? Are they home-made chemically competent or commercial, top10s, omni-maxes, etc.

-blin100-

I am using E. coli XL1B home made competant cells give high efficiency (chemical or electro competant cells).

-christy-

Hi, it's a pity to see your problem, the strtegy you used in your trial is not a good one.
Owing to my experience, the BamH I/Xho I, I have just used it for a cloning and gained the positive one. Here is my advice on your non-positive ones and your possible problems:\

!) As mentioned in Christy and Blin 100 you should set a blank control and negative control if you have tested your competent cells. If not, I suggest you make a transformation effiency test for your competent cells used: the procedure is very easy, diluted your confirmed plasmid at a concentration with 0.1 ng/ul or 1ng/ul, then used 1 ul to take your transformation in 100ul competent cells, and used 900 ul SOC or LB medium for culture~ 45min to 1hr and spreader on the selective plate with 100 ul. So the plate has a 1/10 of your plasmid and calculated clones in whole plate and calculate the transformation efficiency used * clones per ug DNA.ususually 10 6-107 clonies/ug DNA may be used for a common cloning usage. I used my self-prepared competent cells E.coli JM109 for my transformation.

2) It is unwise to use PCR to confirm your ligation efficiency, and unneccessary.

3) The Cloning PCR indicate no positive ones is not equeal to no positive ones. My collegue has crossed such questions before, this not a good method for testing your positive ones. Before your sequencing, it is a well and convincing method is mimiprep your plasmid and make a digestion and this almost nake no false positive. I usually used this method although my collegues dislikes to do so. The prefor the cloning PCR. Cloning PCR definitely has a high false positiveratio.

Anyhow, good luck to you!

-Jason_LuYZ-