SEQanswers - View Single Post - ChIP-Seq: Single-tube linear DNA amplification (LinDA) for robust ChIP-seq.

a3911145 · 06-05-2012, 07:24 AM

I am a PHD student from China
Similar to thedavid, I think I have the same problem in LinDA: very low efficiency of the‘T’ tailing or low efficiency of attaching T7 primer.
I choose another Shrimp alkaline phosphatase produced by Fermentas. I have succeeded in LinDA for only one time, when I add dTTP without ddCTP, and inactivated Klenow at 75° for 20min (start with 50ng DNA [input DNA, fragmented by Ultrasound] produces 17000ng RNA and finally 5000ng DNA). But when repeat the experiment, I failed again and again, with no RNA yield from 20-50ng DNA starting material and no ampification of ChIP-DNA confirmed by realtime PCR.
Working hard on it for over two months, I'm now can confirm that this protocol can work, but I don't understand why it is so hard to stabilize the system!
Has anyone not in Hinrich Gronemeyer's lab gotten this process to work?
Has anyone know why the efficiency of ‘T’ tailing/attaching T7 primer is low?
Thanks a lot!

06-05-2012, 07:24 AM	#9
a3911145 Junior Member Location: China Join Date: Jun 2012 Posts: 2	I am a PHD student from China Similar to thedavid, I think I have the same problem in LinDA: very low efficiency of the‘T’ tailing or low efficiency of attaching T7 primer. I choose another Shrimp alkaline phosphatase produced by Fermentas. I have succeeded in LinDA for only one time, when I add dTTP without ddCTP, and inactivated Klenow at 75° for 20min (start with 50ng DNA [input DNA, fragmented by Ultrasound] produces 17000ng RNA and finally 5000ng DNA). But when repeat the experiment, I failed again and again, with no RNA yield from 20-50ng DNA starting material and no ampification of ChIP-DNA confirmed by realtime PCR. Working hard on it for over two months, I'm now can confirm that this protocol can work, but I don't understand why it is so hard to stabilize the system! Has anyone not in Hinrich Gronemeyer's lab gotten this process to work? Has anyone know why the efficiency of ‘T’ tailing/attaching T7 primer is low? Thanks a lot!