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pcDNA 3.1 Directional TOPO clone issues - (Dec/01/2013 )

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Hello, everyone

I'm fresh here. And I have some problems with pcDNA 3.1 Directional TOPO cloning. I've been trying to insert a 3KB fragment into the pcDNA 3.1 D/V5-His-TOPO vector,then transform the recombinant plasmid into TOP10 competent cells. However, there are no or very few colonies on the plate.

I need all of you experts' instructions. Help me please.

-Jeremy55-

Welcome

 

Details would help - what have you done, how did you prepare the insert?

-bob1-

Welcome,

 

are those few colonies containing the insert you are expecting?

-ostiaziocan-

Welcome

 

Details would help - what have you done, how did you prepare the insert?

Thank you.

After identifying the PCR products, and then performing agarose gel purification, just carried out the ligation and transformation according to the manual.But there were few or no colonies on the plate.

-Jeremy55-

Welcome,

 

are those few colonies containing the insert you are expecting?

Thank you.

It's a small probability event. I need lots of clones to do my subject, and the vector is very expensive. 

-Jeremy55-

 

Welcome

 

Details would help - what have you done, how did you prepare the insert?

Thank you.

After identifying the PCR products, and then performing agarose gel purification, just carried out the ligation and transformation according to the manual.But there were few or no colonies on the plate.

 

How did you amplify (which enzyme)?  Did you have transformation controls?  Did the controls work?

-bob1-

 

 

Welcome

 

Details would help - what have you done, how did you prepare the insert?

Thank you.

After identifying the PCR products, and then performing agarose gel purification, just carried out the ligation and transformation according to the manual.But there were few or no colonies on the plate.

 

How did you amplify (which enzyme)?  Did you have transformation controls?  Did the controls work?

 

PrimeStar HS DNA polymerase. 

Yes, I transformed an Amp+  plasmid into TOP10 cells, and it worked well.

-Jeremy55-

 

 

Welcome

 

Details would help - what have you done, how did you prepare the insert?

Thank you.

After identifying the PCR products, and then performing agarose gel purification, just carried out the ligation and transformation according to the manual.But there were few or no colonies on the plate.

 

How did you amplify (which enzyme)?  Did you have transformation controls?  Did the controls work?

 

Have you used the vector?

-Jeremy55-


PrimeStar HS DNA polymerase. 

Yes, I transformed an Amp+  plasmid into TOP10 cells, and it worked well.

 

That should be fine.  Are you sure you added the required bases to the 5' end of the primer?

-bob1-

 


PrimeStar HS DNA polymerase. 

Yes, I transformed an Amp+  plasmid into TOP10 cells, and it worked well.

 

That should be fine.  Are you sure you added the required bases to the 5' end of the primer?

 

Yes, I'm sure. 

-Jeremy55-
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