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