library frgments size

miraiama · 11-29-2012, 04:29 AM

Hi,
I'm preparing a library using NEB ChIP-seq kit. I'm starting with ssDNA of 250-800nt, making it double stranded using Klenow and random primers and then using the kit. The size of the fragments that I get after the PCR amplification is around 400bp (that includes the 120bp addition of the adapters and primers). I tried to start the library preparation with dsDNA the same size (250-800bp) and got the same 400bp library, so I don't think that the Klenow step is the problem.
Do you have any ideas why do I have selection for the short fragments and how to overcome it (since it is very important to me that the final library will represents all lengths).
Thanks

kwaraska · 11-29-2012, 06:39 AM

I haven't used the NEB chip kit but most kits have a size selection step that would cause other sizes to be removed.

If you skip that step you should get all sizes. When you go to sequence them just remeber to lower the loading concentration a bit as 800 strting will be over 900 bases as a library and takes up quite a bit of real estate on the the flow cell and it is easy to overcluster.

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
determining library size	seqgirl123	Illumina/Solexa	5	07-16-2013 02:43 PM
more counts than total library size ?	Anna Nad	Bioinformatics	5	11-13-2012 02:04 AM
small library size (HiSeq)	Robby	Illumina/Solexa	5	11-23-2011 08:07 AM
TruSeq library size	HGENETIC	Sample Prep / Library Generation	2	11-14-2011 02:56 AM
Expected Library size after adaptors	JPC	SOLiD	3	10-04-2011 07:54 AM

11-29-2012, 04:29 AM	#1
miraiama Junior Member Location: Jerusalem Join Date: Oct 2012 Posts: 1	library frgments size Hi, I'm preparing a library using NEB ChIP-seq kit. I'm starting with ssDNA of 250-800nt, making it double stranded using Klenow and random primers and then using the kit. The size of the fragments that I get after the PCR amplification is around 400bp (that includes the 120bp addition of the adapters and primers). I tried to start the library preparation with dsDNA the same size (250-800bp) and got the same 400bp library, so I don't think that the Klenow step is the problem. Do you have any ideas why do I have selection for the short fragments and how to overcome it (since it is very important to me that the final library will represents all lengths). Thanks

11-29-2012, 06:39 AM	#2
kwaraska Senior Member Location: Boston,MA Join Date: Nov 2008 Posts: 122	I haven't used the NEB chip kit but most kits have a size selection step that would cause other sizes to be removed. If you skip that step you should get all sizes. When you go to sequence them just remeber to lower the loading concentration a bit as 800 strting will be over 900 bases as a library and takes up quite a bit of real estate on the the flow cell and it is easy to overcluster.