Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation

Similar Threads
Thread Thread Starter Forum Replies Last Post
Starting concentration of sample/control in ChIP Seq mnandita Epigenetics 2 12-15-2011 10:02 AM
ChIP-seq DNA concentration jazz Illumina/Solexa 1 10-07-2011 08:16 AM
TruSeq DNA adapters in RNA-seq prep... concentration? ScottC Sample Prep / Library Generation 4 09-10-2011 03:38 AM
Better to risk low concentration library or further amplification? krobison Sample Prep / Library Generation 3 08-12-2010 02:40 PM
Low concentration GA library Melanie Sample Prep / Library Generation 4 08-04-2010 02:34 PM

Thread Tools
Old 09-05-2009, 07:44 PM   #1
Junior Member
Location: Emeryville

Join Date: Sep 2009
Posts: 1
Exclamation Anyone use low concentration for ChIP-seq prep?

Hello All,

I am having great difficulty quantitating my IP samples to use for ChIP-seq. When I used PicoGreen I found that I may only have approximately 4ng of sample. Has anyone used less than 10 ng of starting material for ChIP-seq? Any advice would help at this point!
Audry888 is offline   Reply With Quote
Old 10-19-2009, 06:41 AM   #2
Location: Vienna

Join Date: Mar 2008
Posts: 45

We have prepared ChIP-Seq samples with as low as 1.5ng total amount and have obtained good results.
Make sure material is in the right size range so you can recover most of it out of the gel.

andibody is offline   Reply With Quote
Old 10-19-2009, 01:33 PM   #3
Junior Member
Location: California

Join Date: Sep 2008
Posts: 2
Default ChIP-seq sample prep. with low input

Hello Andibody,

I just read your post and wanted to ask if you diluted the adaptors, and assuming so, to what degree. I have been working on generating libraries starting with low ChIP DNA input, and have some success, but measured thus far. I've managed to successfully PCR up the gel isolates, but there is evidence of what I suspect are PCR products resulting from concatamerization. I intend to isolate the 2 major PCR products by repeating a gel fractionation/extraction followed by a round of PCR, followed by, cloning verification if needed to determine which of the two species is correct.

Any thoughts?

Best regards,

C.Griffin is offline   Reply With Quote
Old 10-29-2009, 04:33 AM   #4
Location: Vienna

Join Date: Mar 2008
Posts: 45

sorry for the late answer. We diluted adaptors 1:20 but I suspect that we could even go up with the dilution. We have observed a second band double the size of the expected fragments although but we are not sure where it comes from (seems as nobody really knows yet, there are some threads on this issue). But, to my experience, this observation is not related to low starting amouts of sample.
If the bigger band is small, we add it up to quantification and sequenced all material. If it is bigger than let's say 1/3 of the 'right' band, we perform a second size selection. Both ways have given good results, nonetheless, we are still afraid of bringing any bias into our sample when this kind of artifacts show up.

andibody is offline   Reply With Quote
Old 05-28-2010, 12:37 PM   #5
Junior Member
Location: Florida

Join Date: Nov 2009
Posts: 4

Hi guys,
I am happy to read your valuable discussion here. We recently had problem that the color balance of the sequencing is completely off, which caused majority of image pannel failed. On the WFA, every parameter looks fine except P2_Gain is too low (lower than 8). Is that color balance off is caused by the concatamerization of adaptors? We did not know this trick and did not dilute the adaptors while our ChIP DNA concentration is very low.

Thanks in advance.

smallcreek is offline   Reply With Quote

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off

All times are GMT -8. The time now is 06:17 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2022, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO