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ta cloning problem


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#1 deespike

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Posted 02 November 2010 - 07:09 AM

Hi all

I have trying to clone a 1kb gene into a ta cloning vector pTZR/T (fermentas)2.8 kb. I did blue white selection and got both type of colonies but when i screened white colonies they were all negative only but when compared with the control i.e. plasmid isolated from the blue colony it showed considerable shift.

I donot understand how there can be shift in the plasmid yet not having any insert.

Plz help.

#2 perneseblue

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Posted 02 November 2010 - 06:05 PM

Did you use restriction digests to screen the candidate colonies?

Could you please elaborate by "shift in the plasmid". It is a bit unclear.

Did you gel purify the 1kb insert? Could you please provide more details on how this ligation was conducted.
May your PCR products be long, your protocols short and your boss on holiday

#3 lsek

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Posted 05 November 2010 - 01:22 PM

Hmmm....

I'm guessing "shift in the plasmid" means the band size difference of plasmid DNA of white colony as compared to blue colony, as observed in the agarose gel electrophoresis. If that is the case, I wouldn't define it as "no insert".

Hope you have got it fixed.

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