Truseq DNA PCR-free kit

Laruo · 01-27-2014, 08:12 AM

Hi!

I´m initializing in NGS library construction. I will start with Truseq DNA PCR-free kit and I would like to ask you a couple of details. I know people that, following the old protocol runs a Bionalyzer chip after the DNA fragmentation. But in the new protocol, where you have two options for the fragmentation conditions depending on your sequencing purposes, I read you have a cleaning step to be done immediately after the fragmentation, followed by the end repair and size selection steps. Does anyone run the Bioanalyzer in this case?

Based on your experience which are the steps where one could stop the protocol for a few days?

thanks!

03-05-2014, 03:42 AM

Hi Laruo,

I think the reason why they don't suggest a Bioanalyzer after fragmentation is because they optimized the protocol for two specific insert sizes - 350 and 550 bp.

If your DNA is good, there shouldn't be any problems with the fragmentation if you use the parameters that they suggest. If you are still unsure about it, I think you can just take 1 µl of sheared DNA (of the 52.5 µl from the Covaris tube) and run it on a HighSensitivity Bioanalyzer chip.
On general, you can always test your shearing beforehand by not doing an Illumina library, but just shearing your DNA and then checking it with the Bioanalyzer or a normal gel.

Illumina mentions all the safe stopping points in their protocol where you can freeze (-20ºC) the library and continue some other day:
1) after end repair and size selection
2) after adapter ligation
3) after PCR
be sure to perform the necessary clean-up steps before you freeze the library.

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01-27-2014, 08:12 AM	#1
Laruo Member Location: Spain Join Date: Feb 2012 Posts: 13	Truseq DNA PCR-free kit Hi! I´m initializing in NGS library construction. I will start with Truseq DNA PCR-free kit and I would like to ask you a couple of details. I know people that, following the old protocol runs a Bionalyzer chip after the DNA fragmentation. But in the new protocol, where you have two options for the fragmentation conditions depending on your sequencing purposes, I read you have a cleaning step to be done immediately after the fragmentation, followed by the end repair and size selection steps. Does anyone run the Bioanalyzer in this case? Based on your experience which are the steps where one could stop the protocol for a few days? thanks!

03-05-2014, 03:42 AM	#2
lorendarith Guest Posts: n/a	Hi Laruo, I think the reason why they don't suggest a Bioanalyzer after fragmentation is because they optimized the protocol for two specific insert sizes - 350 and 550 bp. If your DNA is good, there shouldn't be any problems with the fragmentation if you use the parameters that they suggest. If you are still unsure about it, I think you can just take 1 µl of sheared DNA (of the 52.5 µl from the Covaris tube) and run it on a HighSensitivity Bioanalyzer chip. On general, you can always test your shearing beforehand by not doing an Illumina library, but just shearing your DNA and then checking it with the Bioanalyzer or a normal gel. Illumina mentions all the safe stopping points in their protocol where you can freeze (-20ºC) the library and continue some other day: 1) after end repair and size selection 2) after adapter ligation 3) after PCR be sure to perform the necessary clean-up steps before you freeze the library.