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epi123's Content

There have been 8 items by epi123 (Search limited from 23-February 19)


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#86224 ChIP library prep for TF - ideas?

Posted by epi123 on 08 September 2010 - 06:53 AM in ChIP and Next Generation Sequencing

What is the minimum amount of material you can use going into illumina library? Solid ChIP-Seq protcol uses Magnify chip with library protocol for samples less than 10ng.



#64313 DNA sonication - RNA contamination?

Posted by epi123 on 26 March 2010 - 05:51 AM in ChIP and Next Generation Sequencing

happy to see you got your profile. keep in mind if your are planning on sequencing, that chelex resin generates too much ssDNA...really difficult to get enough dsDNA for library preparation.



#63665 DNA sonication - RNA contamination?

Posted by epi123 on 23 March 2010 - 07:36 AM in ChIP and Next Generation Sequencing

looking over the MAGnify manual.. it says to use 1million cells per 50ul Lysis buffer. What's the reason to lyse in IP buffer without SDS?
Thanks



#63131 DNA sonication - RNA contamination?

Posted by epi123 on 18 March 2010 - 06:14 AM in ChIP and Next Generation Sequencing

I'm not used to this sonicator. But how many cells were you starting with? How much lysis buffer?



#62070 Suitable antibody for H3K4me3?

Posted by epi123 on 10 March 2010 - 11:16 AM in ChIP and Next Generation Sequencing

other source for H3-K4Me3 antibody- Invitrogen, Epigentek



#58669 First ChIP - good start?

Posted by epi123 on 12 February 2010 - 07:19 AM in ChIP and Next Generation Sequencing

Mighty Mouse is correct in terms of pol2 enriching to regions where the mechanisms are still unclear, like ncRNA. you may also consider titrating the antibody although your data looks great. alternatively, 2hrs incubation could be cut back to 1hr. :-)



#53947 Protein G vs Protein A for Rabbit Antibodies

Posted by epi123 on 05 January 2010 - 08:06 AM in ChIP and Next Generation Sequencing

my understanding is that protein A beads are generally better suited for rabbit polyclonal antibodies. i've seen the mouse monoclonals enrich better with protein G. Years ago i would use protien a/g sepharose beads but lately i'm using the MAGnify kit from invitrogen that uses a mix of protein a/g magnetic beads



#43202 Suitable antibody for H3K4me3?

Posted by epi123 on 07 November 2009 - 10:20 AM in ChIP and Next Generation Sequencing

My original choice was abcam's ab8580, but this is out of stock and their staff say they have no idea when new batches will be available. Upstate/Millipore's 07-473 is also now out of stock, and I'm waiting on their customer services to let me know if more will be available.

These two antibodies are by far the most popular I've seen in ChIP experiments. I will be using ChIP DNA for next gen sequencing so it's absolutely critical the antibody works well. Does anyone have a suggestion for an alternative, that has been used in a high profile publication, or you have personal experience with?

Thanks.



Invitrogen and Cell signalling have this antibody as well but don't know any publications.




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