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I have cloning problems with pGEM-T Easy - (May/06/2010 )

I do PCR, aim for amplify NRPS gene form soil.

I use goTaq® polymerase.

PCR product has many bands, but contain band with size of interest.

I select the PCR product which has 300 bp in size by gel electrophoresis and kit extraction.

The PCR product is dissolved in distilled water.

I ligate it into pGEM-T Easy vector, incubate in refridge (app. 5 degree celcius) for 2-3 days.

Then i electroporate the pGEM-insert DNA into DH5a.

The results show no colony. I'm sure that transformation is ok, since my plasmid (got them from blue colony containing pGEM vector inside)

can be transformed and grow well on LB ampicillin Xgal IPTG plate.

(Troubleshooting in user manual says that it may has some problem in transformation)

Also, my positive control (control DNA for ligation control from pGEM-T Easy vector system) cannot survive the electroporation.

I'm struck in this step for several months. Please help me.

Thanks for your advance.

-Placebosz-

Placebosz on May 5 2010, 11:41 PM said:

I do PCR, aim for amplify NRPS gene form soil.

I use goTaq polymerase.

PCR product has many bands, but contain band with size of interest.

I select the PCR product which has 300 bp in size by gel electrophoresis and kit extraction.

The PCR product is dissolved in distilled water.

I ligate it into pGEM-T Easy vector, incubate in refridge (app. 5 degree celcius) for 2-3 days.

Then i electroporate the pGEM-insert DNA into DH5a.

The results show no colony. I'm sure that transformation is ok, since my plasmid (got them from blue colony containing pGEM vector inside)

can be transformed and grow well on LB ampicillin Xgal IPTG plate.

(Troubleshooting in user manual says that it may has some problem in transformation)

Also, my positive control (control DNA for ligation control from pGEM-T Easy vector system) cannot survive the electroporation.

I'm struck in this step for several months. Please help me.

Thanks for your advance.

maybe it isnt your electroporation, some times after gel extraction 3a overhangs are removed so you can add terminal a after gel extraction by tag and then ligate it, and by the way we get good results by cacl2 transformation why dont you try it?

-sara.r-

So if i'm right: neither your control or your pGEMTeasy-ligation is growing?
Then i think it might be a problem from transformation. I'm also wondering why you electroporate? A heatshock should give enough colonies and is much simplier. When you electroporate do you give your bacteria SOC-medium after the pulse? This way they recover faster and more chance on living bacteria.
Good luck

-fysio lab-

Hi,
I agree that probably the transformation is a problem
and I also recommend to do transformation using CaCl2.
Why did you store your ligation mixture for so long at 5C?
When your ligation is finished you can just keep it at -20C.

-Agnes85-

If you perform a transformation you can always do a control -> put a little bit of bacteria on a separate plate without selection marker -> a lot of colonies should grow if bacteria survived the transformation.

-Agnes85-

Thanks to everyone

I tried heatshock, but CaCl2, i didn't.

My friend say like sara.r opinion, i'll try today. Thanks for your suggestion.

To Fysio lab, yes, neither of my transformation control or ligation control give me a colony when i did heat shock.

but when i do electroporation, my transformation control give me some colony.

However, my white colony from that reaction are lesssssssssssss than blue ones, and they usually don't contain insert.

Agnes85, my lab partner found that when he ligate his reaction for 2-3 days at refrige temp. He get more clone than overnight incubation.

(i do it for incubation, not storage)

Thanks for suggestion of "put a little bit of bacteria on a separate plate without selection marker" i'll do it this time.

Again, thank you very much.

Sorry for reply all of you so late.

-Placebosz-