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No band after digestion - (Aug/11/2010 )

Hi,

I was wondering if anyone could help me. I have tried to clone 240 bp PCR product cutting with NdeI/XhoI (restriction site in the primer) into pET23a+ cut with the same enzyme for a long time. There was no positive clone after many times cloning. After changing the host strain to K12, there were clones and then I extracted the plasmid with 5 PRIME kit and double digested with NdeI/XhoI. After running the gel, plasmid band was found but no or empty band in the digested plasmid. The control pET23a cut with NdeI/XhoI showed correct band. These showed no error in water, RE , buffer or BSA). Please help me to clarify this.

Thanks in advance

-Pig-

Hello :)

How are you purifying your PCR product?

Clare

Pig on Wed Aug 11 09:30:22 2010 said:


Hi,

I was wondering if anyone could help me. I have tried to clone 240 bp PCR product cutting with NdeI/XhoI (restriction site in the primer) into pET23a+ cut with the same enzyme for a long time. There was no positive clone after many times cloning. After changing the host strain to K12, there were clones and then I extracted the plasmid with 5 PRIME kit and double digested with NdeI/XhoI. After running the gel, plasmid band was found but no or empty band in the digested plasmid. The control pET23a cut with NdeI/XhoI showed correct band. These showed no error in water, RE , buffer or BSA). Please help me to clarify this.

Thanks in advance

-Clare-

When you prep the plasmid did you make sure the ethanol was added in PE ?

-ranvi-

Hi,

Thanks for the reply. I was away due to long holiday here.

I used the Nucleospin kit to purify the PCR product.

I was absolutely sure that I added EtOH to the PE.

What happened?

-Pig-

Pig on Mon Aug 16 05:28:32 2010 said:


Hi,

Thanks for the reply. I was away due to long holiday here.

I used the Nucleospin kit to purify the PCR product.

I was absolutely sure that I added EtOH to the PE.

What happened?

I have the same problem.
What heppend in the end? How you solved it?

-sani-