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Probe Hybridization

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#1 nadim999



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Posted 10 August 2004 - 04:56 AM

I am trying to screen a library consisting of genomic insert in pUC18 plasmids and transformed in DH5a E coli.

After lifting the colonies on nylon membrane, I am screening them with a repetitive probe of T(AG)8. I have also included a positive control (a clone consisting of T(AG)8 insert in pUC18).

I am using Church Buffer for pre-hyb and hyb @42C. And for stringency washes I am using 2xSSC 0.1% SDS @ rtp for 5 mins on the first wash followed by 0.5xSSC 0.1%SDS. @ 48C for 15 mins on the second wash.

The problem is I am not getting any signals, not even for my positive control.

Is there any suggestions? How good is Church buffer for hybridization?
What temperature should the hybridization and stringency wash should follow?

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