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No band after digestion


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4 replies to this topic

#1 Pig

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Posted 11 August 2010 - 01:30 AM

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

Edited by Pig, 11 August 2010 - 01:53 AM.


#2 Clare

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Posted 11 August 2010 - 03:48 AM

Hello :)

How are you purifying your PCR product?

Clare

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



#3 ranvi

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Posted 11 August 2010 - 05:16 AM

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

#4 Pig

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Posted 15 August 2010 - 09:28 PM

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?

#5 sani

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Posted 16 November 2010 - 10:13 AM

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?




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