SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
Optimal concentration of DNA for clustering jecrc Illumina/Solexa 21 02-04-2012 10:14 AM
Starting concentration of sample/control in ChIP Seq mnandita Epigenetics 2 12-15-2011 09:02 AM
ChIP-seq DNA concentration jazz Illumina/Solexa 1 10-07-2011 07:16 AM
concentration of my starting material? seqgirl123 Illumina/Solexa 6 07-28-2009 06:19 PM
PubMed: Profiling DNA methylation from small amounts of genomic DNA starting material Newsbot! Literature Watch 0 11-07-2008 09:30 AM

Reply
 
Thread Tools
Old 06-19-2012, 07:29 AM   #1
balaji_chattopadhyay
Junior Member
 
Location: India

Join Date: Jun 2012
Posts: 3
Default help regarding starting concentration of dna

Hi,
This is a problem I have stated elsewhere as well. I am working with genomic DNA of mammals. I am trying to standardize RADTAG protocol. In this regard I need to know the importance of genomic DNA concentration for restriction digestion and the least quantity of genomic DNA that can be used. I use Qiagen DNA extraction kit and my samples have typically shown ~ 10ng/ul concentration in nanodrop. But flourometer (Qubit), returned genomic DNA concentration of~2ng/ul. The radtag protocols available usually refers to starting quantity of 100ng to 1ug, with most reports suggesting 1ug. At the most I can try to concentrate my DNA and may obtain ~50 to 100 ng yield for a 50ul restriction digestion experiment.
It would help me much if I can get some advice regarding possibilities of standardization. My samples are obtained through non lethal sampling of wild caught animals and it may not be possible to obtain more tissue or repeat sampling.
__________________
Balaji
balaji_chattopadhyay is offline   Reply With Quote
Old 06-21-2012, 09:35 AM   #2
bbentlage
Junior Member
 
Location: MD USA

Join Date: Jun 2012
Posts: 1
Default

Hey there!

I haven't done RAD tags myself but I have two comments that come immediately to mind.

1) Personally, I would trust the Qbit more than the Nanodrop considering how concentrations are measured i the two different technologies.

2) If you must "make" more DNA and cannot get more tissue samples you could look into something like the Genomiphi kit. Essentially you amplify the DNA in your sample using random hexamers and a proof-reading tag.

-Basti

Last edited by bbentlage; 06-21-2012 at 09:37 AM.
bbentlage is offline   Reply With Quote
Reply

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 05:19 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO