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Colony lift hybridisation positive but clones are negative for insert DNA

Cloning Colony lift Hybridisation

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#1 Rara

Rara

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Posted 31 January 2012 - 03:15 PM

Hi,
We have subcloned a gene of interest into a vector and followed the appropriate protocols to transform competent cells etc. we initially selected 26 colonies for mini prep and restriction digest analysis to identify positive colonies. This proved to be unsuccessful. We therefore went to our usual backup plan of colony lifts and probe hybridisation to identify positive colony. To our delight, we got a number of 'hotspots'. We selected the colonies for confirmation by RE digest. Unfortunately, they turned out to be negative and to be empty vector. I can't understand why our hybridisation screen gave positive signals but the colonies were infact empty vector. The colony lift did use a plate that was slightly old but I still can't see why this would result in a positive signal. Control probes etc worked well.. Any ideas? Will repeat but intrigued for an answer.

#2 akhshik

akhshik

    Nice day for research

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Posted 03 February 2012 - 10:26 PM

hello there,
possible answers:

1) your Probe designed to detect you vector not the gene of interest
2) your cell has lost you gene of interest
3) your mistake in experiment

Check the first one first!

cheers,
Akhshik.





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