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Cloning a big insert in a small vector


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#1 PV_effect

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Posted 05 February 2015 - 10:50 AM

Hi everyone,

I had some issues with a cloning since a couple of months. I explain: I want to insert a ~6000pb gene into a mamalian plasmid. The PCR from cDNA went well I have my insert (even if it's not really a thight band) and I purify it using a Qiagen kit. then I digested the insert and the vector with BglII/NotI and proceed to the ligation. I got some colonies but everytime that I made some analytical digestion to confirm it I don't have the right "size".

So I tried the TA cloning and no colony @all....

 

So I tried ro change the strain DH5alpha, TOP10 and pirHC, the growth temp (37 and 30°C).. and each time nothing (I mean no positive clone);

So I tried to just ligate directly into the expression vector and I got the same things.....

 

Did someone have an idea to sucess?

Thank you

 

 



#2 labtastic

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Posted 05 February 2015 - 11:37 AM

Have you tried blunt ended TOPO cloning? Might be worth a try. 

 

Is your PCR from cDNA pretty clean, i.e. a single band? If so, then consider instead of doing a gel purification, just add your PCR rxn directly to the TOPO cloning reaction. Screen for correct colonies with colony pcr. Or, just PCR cleanup your pcr reaction and add that directly to the TOPO rxn. The reason I suggest not doing the gel purification is because inserts that big can be more prone to UV damage while you are cutting our your gel slice. 

 

If your PCR reaction is not particularly clean, how are you doing the PCR? Do touchdown PCR if you aren't already. A friend from a lab across campus introduced me to it a few years ago and I've never gone back. PCR's are now so much cleaner.

 

On an unrelated note (or maybe related), for the last 6-8 months our lab had a lot of trouble with qiagen kits. One day all the cloning the lab just went to hell. Everybody was struggling to get basic things to work. We re-ordered new kits and still the same troubles. We have since switched everything over to different company and cloning is working well again. If a lab down the hall uses a different system than qiagen, ask them if you can use their kit to do the cleanup work.

 

Just some ideas is all.

 

Good luck.  



#3 PV_effect

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Posted 06 February 2015 - 10:42 AM

Hi,

Actually my PCR product is a clear clean band even if the yield is low. But thank you for the tricks :).

I will try the blunt end TOPO, hope that it will be OK

 

Actually we had some cloning issues to since november in all the lab, we will try to see if it's because of the kit (which will be a real pain).

 

Thanks a lot






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