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pSuper cloning problems


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#1 clarem

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Posted 08 May 2009 - 01:24 AM

Hi,
Just wondering if anyone has any experience of cloning shRNA into the pSuper vector?
I am having great difficulties in doing so!
When I digest the plasmid with bglII and hindIII, I am still getting a large amount of colonies on my no insert with and without ligase control plates. This is despite repeating the digest on the same sample 2-3 times, and digesting overnight each time with bglII. Other people have been using these enzymes in the lab and so it is not a problem with the enzymes.
I have used a range of different vector:insert ratios but any colonies I get have all been negative for my insert, when I perform a HindIII/EcoRI digest check.
Does anyone have any ideas of what could be happening, and any suggestions of what I can do next?
Would CIP-treating the vector have any benefit?
This is beginning to drive me insane :D
Thanks for any help :)

#2 T C

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Posted 08 May 2009 - 02:57 AM

Hey,

I think the vector concentration that you are using for digestions is too high, use lower concentrations and setup multiple digests. Cipping shoudl help if you have single digests.

Best,
TC

Hi,
Just wondering if anyone has any experience of cloning shRNA into the pSuper vector?
I am having great difficulties in doing so!
When I digest the plasmid with bglII and hindIII, I am still getting a large amount of colonies on my no insert with and without ligase control plates. This is despite repeating the digest on the same sample 2-3 times, and digesting overnight each time with bglII. Other people have been using these enzymes in the lab and so it is not a problem with the enzymes.
I have used a range of different vector:insert ratios but any colonies I get have all been negative for my insert, when I perform a HindIII/EcoRI digest check.
Does anyone have any ideas of what could be happening, and any suggestions of what I can do next?
Would CIP-treating the vector have any benefit?
This is beginning to drive me insane :D
Thanks for any help :)






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