I prepared a couple ChIP-seq libraries using the exact protocol from the Illumina ChIP-seq library prep kit. Except at the last step of library enrichment by PCR I dived each sample in half. Half I amplified with Phusion polymerase and the other half I amplified with the Kapa Biosysmtems HiFi Library Amplification polymerase.
The cycling conditions were as follows:
Initial denaturation: 45 sec 98˚ C
18 cycles of: 15 sec 98˚ C
30 sec 65˚ C
30 sec 72˚ C
Final extension: 1 min 72˚ C
1 ul of product was run on the Agilent Bioanalyzer (DNA 1000 kit).
With the Kapa Biosystems polymerase all the DNA is up between 500 and 700 bp.
With Phusion polymerase most of the material is in the expected range between 200 and 300 bp. Although there is some up in the 500 to 700 bp range.
The starting material was digested with Micrococcal Nuclease so that about 80% of the DNA was mono-nucleosomal (145bp). Before library amplification the DNA was gel purified according the the instructions of the Illumina kit.
What is causing the large DNA fragments? How can it be minimized?
Kapa technical services says it is probably due to over amplification. Why would over amplification cause the fragments to double in size?
Anybody else using Kapa HiFi polymerase for ChIP-seq library amplification? How many cycles are you using?
Any suggestions greatly appreciated.
The cycling conditions were as follows:
Initial denaturation: 45 sec 98˚ C
18 cycles of: 15 sec 98˚ C
30 sec 65˚ C
30 sec 72˚ C
Final extension: 1 min 72˚ C
1 ul of product was run on the Agilent Bioanalyzer (DNA 1000 kit).
With the Kapa Biosystems polymerase all the DNA is up between 500 and 700 bp.
With Phusion polymerase most of the material is in the expected range between 200 and 300 bp. Although there is some up in the 500 to 700 bp range.
The starting material was digested with Micrococcal Nuclease so that about 80% of the DNA was mono-nucleosomal (145bp). Before library amplification the DNA was gel purified according the the instructions of the Illumina kit.
What is causing the large DNA fragments? How can it be minimized?
Kapa technical services says it is probably due to over amplification. Why would over amplification cause the fragments to double in size?
Anybody else using Kapa HiFi polymerase for ChIP-seq library amplification? How many cycles are you using?
Any suggestions greatly appreciated.
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