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Large vector cloning-recombination problem?


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#1 ApeII

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Posted 11 September 2010 - 07:26 AM

Hi Everyone,

I'm cloning a 3kb right arm into a 13kb targeting vector (pBR322 ori), using a single enzyme SalI. I could obtain hundreds of colonies but no one contains insert from 40 colonies I've screened. The undigested DNA of most colonies contain a vector band and a foreign band.
My background(vector only)has only several colonies.
Is it a recombination problem? I've tried Top 10, DH5a.
Thanks for your help in advance.

ApeII

#2 perneseblue

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Posted 16 September 2010 - 08:34 PM

SalI claims another victim!

Perhaps a bit over dramatic but for reasons unknown, ligation using SalI restriction sites often encounter problems. I have learned to avoid using SalI. My immediate reaction would be use some other enzyme.

But that being said, there isn't enough details in your post to actually determine what (if anything) has gone wrong. Could you please provide every detail of how this ligation was done. What was digest mix, how long was the DNA cut. How was the 3kb insert made. If by PCR, how many bp was added around the restriction site at the 5' end of the primer. Was the DNA fragment gel purified? How long was the UV exposure. What was the ligation mix? Is your T4 DNA ligase still good? Is this the only ligation facing problems? Does the t4 ligase buffer still have a strong smell? How was the DNA transformed? Did you run the ligation mix on a gel to check to make sure that there was DNA ligated and present just prior to transformation.
May your PCR products be long, your protocols short and your boss on holiday




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