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Old 07-26-2012, 01:57 PM   #14
MolBioAsk
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Location: Philadelphia

Join Date: Jul 2012
Posts: 1
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Hi,

I am struggling with LinDA now, and thanks for all your helpful suggestions and ideas on the subject, especially from Linampli.

I used my ChIP pulled down chromatin 2ng(maybe nanodrop not acurate) to do LinDA and got specific amplification, total amount 180ng; and when I use 5opg of pulled down DNA for LinDA, I got low yield non-specific amplification.I think I should start with accurate 50pg of Input DNA to set up the experiment first.

Here are my questions:
1. 50pg of sonicated chromatin(100bp to 1kb) and 50pg of 250bp DNA fragment is different. Did someone get ideal amplification from 50pg of sonicated chromatin?

2. Several post above said they got lower efficiency of T-tailing and oligo T7-annealing. how to improve the efficiency, other than usage of TSAP, eg, increase reaction time, increase the enzyme de-activation temparature..or..?

Hope to get some help here. Thanks a lot.
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