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Forum: Sample Prep / Library Generation 08-15-2016, 04:14 PM
Replies: 1
Views: 819
Posted By Hamid
Hi Shldbcrzy1, can you please provide a few...

Hi Shldbcrzy1,

can you please provide a few details of your workflow:
1. are you cryofrcturing the fixed tissue as advised for tissue ChIP?
2. what starting mass ranges of tissue are you working...
Forum: Sample Prep / Library Generation 02-03-2016, 01:29 PM
Replies: 17
Views: 8,345
Posted By Hamid
Physical shearing is better since it is...

Physical shearing is better since it is concentration independent, isothermal, easily controllable as compared to chemical methods, and you don't have to deal with the stringent cleanup methods...
Forum: Sample Prep / Library Generation 02-19-2015, 11:10 AM
Replies: 2
Views: 1,029
Posted By Hamid
Please check the integrity of your DNA samples by...

Please check the integrity of your DNA samples by running some unsheard samples on an agarose gel. Also please note that bioanalyzer chips are very sensitive to contaminants. How were these DNA...
Forum: Sample Prep / Library Generation 01-04-2015, 12:58 PM
Replies: 4
Views: 1,454
Posted By Hamid
Covaris shearing of DNA is indeed concentration...

Covaris shearing of DNA is indeed concentration independent. I have attached one customer's data in which they carried out a shearing of 1-50ug of DNA in 130ul volume in a microTUBE.
As...
Forum: Sample Prep / Library Generation 01-04-2015, 12:44 PM
Replies: 4
Views: 2,349
Posted By Hamid
Hi, Since tagmentation does have an inherent...

Hi,

Since tagmentation does have an inherent insertion bias toward certain preferred sequences, it cannot be considered a random fragmentation system. The other issues with tagmentation are:
1....
Forum: Sample Prep / Library Generation 06-03-2014, 01:01 PM
Replies: 73
Views: 41,113
Posted By Hamid
Hi, There are several possible causes for...

Hi,

There are several possible causes for what you are noticing:
1. over-fixation. We have processed quite a bit of liver tissue for ChIP, and we found that 5 minute fixation is quite sufficient...
Forum: Bioinformatics 03-20-2014, 01:33 PM
Replies: 35
Views: 26,522
Posted By Hamid
Thank you very much GenoMax...It worked great. ...

Thank you very much GenoMax...It worked great.

Is it possible to have the commands be universally available from any directory without having to specify the path?

Thank you

hamid
Forum: Bioinformatics 03-18-2014, 07:28 AM
Replies: 35
Views: 26,522
Posted By Hamid
installing samtools on mac

Hi,

Does anyone have a guide on how to properly install samtools version 0.1.19 on a macbook pro with osx 10.9.2.
I would greatly appreciate your help.

Thank you.

Hamid
Forum: Epigenetics 12-05-2013, 02:27 PM
Replies: 5
Views: 5,053
Posted By Hamid
chromatin shearing does generate a distribution,...

chromatin shearing does generate a distribution, and the partially sheared material serve as better epitopes than the shears material. Hence you are you pulling down larger fragments that what you...
Forum: General 12-05-2013, 02:21 PM
Replies: 12
Views: 9,468
Posted By Hamid
I would not advise reusing of the microTUBEs for...

I would not advise reusing of the microTUBEs for several reasons:
1. glass has an inherent binding capacity for DNA, so contamination will be a significant risk
2. Although there is a well over a 1...
Forum: Sample Prep / Library Generation 04-08-2013, 06:38 PM
Replies: 4
Views: 1,553
Posted By Hamid
Hi Ashely, As we discussed on the phone, you...

Hi Ashely,

As we discussed on the phone, you are:
1. using too many cells for the microTUBE. The microTUBE is designed for only processing nuclei from 3 million cells or less in the 130ul volume....
Forum: Sample Prep / Library Generation 01-30-2013, 12:28 PM
Replies: 4
Views: 2,985
Posted By Hamid
Hi Brian, What you are noticing is due to...

Hi Brian,

What you are noticing is due to one of the following reasons:
1. over-fixed chromatin. when chromatin is over-fixed areas with high protein content tend to over-fix and is quite...
Forum: Epigenetics 01-04-2013, 02:48 PM
Replies: 13
Views: 5,194
Posted By Hamid
Hi Nfourier, You are over-fixing your cells....

Hi Nfourier,

You are over-fixing your cells. I have yet to find a single mammalian cell that requires more than 5 minutes of cross linking at room temperature.
May I suggest that you follow all...
Forum: Epigenetics 12-20-2012, 09:25 AM
Replies: 13
Views: 5,194
Posted By Hamid
Hi nfourier, Simply increasing the...

Hi nfourier,

Simply increasing the processing energy will not work. It will just destroy the epitopes.
Please process your samples according to the Covaris protocol...
Forum: Epigenetics 12-18-2012, 11:32 PM
Replies: 13
Views: 5,194
Posted By Hamid
Hi Nfourier, It is the lane...

Hi Nfourier,

It is the lane number-processing time, so 2-10 means lane 2, 10 minute processing time.
Just using the shearing buffer might not work. You will have to use the truChIP shearing kit...
Forum: Epigenetics 12-18-2012, 11:14 AM
Replies: 13
Views: 5,194
Posted By Hamid
Hi nfourier, Most protocols and reagents...

Hi nfourier,

Most protocols and reagents used today for ChIP were developed and optimized for use with probe or bath sonicators. Since Covaris technology is quite different, different sample...
Forum: Sample Prep / Library Generation 11-01-2012, 10:18 AM
Replies: 5
Views: 2,586
Posted By Hamid
Hi Amed, The best way to place your sample...

Hi Amed,

The best way to place your sample inside the Covaris micrTUBEs is to go through the septa with a p200 pipette tip. Once inside the tube, you then angle the tip slightly such that it is...
Forum: Sample Prep / Library Generation 10-19-2012, 11:55 AM
Replies: 7
Views: 2,194
Posted By Hamid
Hi Harold, I do work for Covaris as a...

Hi Harold,

I do work for Covaris as a scientist, but my post on seqanswers are not sales or marketing related as I do not have an interest in that capacity. I do have an interest in the science...
Forum: Sample Prep / Library Generation 10-18-2012, 10:57 AM
Replies: 7
Views: 2,194
Posted By Hamid
Hi Rocketknight, While the the enrichment...

Hi Rocketknight,

While the the enrichment step is a possible culprit, there is some merit to the shearing methodology used generating a bias in the library preparation. traditional sonicator...
Forum: Sample Prep / Library Generation 08-17-2012, 10:08 AM
Replies: 15
Views: 11,212
Posted By Hamid
Hi wz2145, The settings used is too much for...

Hi wz2145,

The settings used is too much for the 6x16mm microTUBEs. The reason why you are not getting the gradual reduction in shearing size range is that you are over-fixing your cells. Since...
Forum: Sample Prep / Library Generation 06-27-2012, 05:05 PM
Replies: 1
Views: 1,793
Posted By Hamid
Hi LAH, The Covaris M220 is a new personal...

Hi LAH,

The Covaris M220 is a new personal DNA fragmentation system launched at the AGBT this year.
I think it will be a great idea for you to evaluate the M220 in your lab with your DNA samples....
Forum: Sample Prep / Library Generation 06-21-2012, 08:46 AM
Replies: 15
Views: 11,212
Posted By Hamid
Hi Zhou, Sorry for the delay in replying to...

Hi Zhou,

Sorry for the delay in replying to your post about RNA fragmentation. If you are using the Agilent small RNA chip, you will not see the shearing progress. Please use either the total RNA...
Forum: Epigenetics 05-25-2012, 01:41 PM
Replies: 19
Views: 14,114
Posted By Hamid
Hi MGineste, Thank you for that link and...

Hi MGineste,

Thank you for that link and data. While your theory might be plausible for people who do ChIP with carrier DNA, quite a few people including my lab, do not use carrier DNA or DNA...
Forum: Sample Prep / Library Generation 05-25-2012, 01:20 PM
Replies: 4
Views: 2,683
Posted By Hamid
Hi Phillip, There are several possible...

Hi Phillip,

There are several possible reasons for tubes breaking during processing on a Covaris instrument:
1. the Covaris microTUBEs are rated for use with a maximum duty cycle of 10% and...
Forum: Epigenetics 05-22-2012, 01:43 PM
Replies: 19
Views: 14,114
Posted By Hamid
Hi rahulr1485 and MGineste, What you are...

Hi rahulr1485 and MGineste,

What you are noticing is unfortunately an ideal example of epitope destruction during high energy chromatin shearing. What is occurring is that you are over-shearing...
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