SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
low dna concentration of IP samples jon123 Sample Prep / Library Generation 0 07-08-2015 09:02 PM
Low DNA concentration from environmental samples mariana_erasmus Introductions 1 03-24-2015 04:12 AM
Illumina TruSeq PCR-Free - Low Final Concentration WGS Rat DNA Libraries agileta Illumina/Solexa 1 11-18-2014 08:11 PM
Wang, 'Low Cost Library Construction...': Low Concentration MAdkisson RNA Sequencing 2 02-19-2014 03:33 PM
Low concentration DNA, quantity needed and any ideas?! firefly2280 Sample Prep / Library Generation 12 11-05-2012 05:35 AM

Reply
 
Thread Tools
Old 08-11-2015, 06:19 AM   #1
cngl
Junior Member
 
Location: Turkey

Join Date: Aug 2015
Posts: 4
Default Low DNA concentration

My samples are infected teeth and i got my samples cryogenically powdered. I want to extract DNA from these samples and then use pyrosequencing analysis, however there are some issues. The company which will serve 454 Pyrosequencing procedures requires at least 50 ng/無 concentration. In our last call they said if i get higher than 5-10 ng they will perform lineer amplification. But i could not increase DNA.
In order to increase concentration, we elongated our incubation times from 30 to 60 minutes both at 37蚓 and 56蚓. In every 15 minutes i vortexed samples and put back in bath. But the concentration values did not change. I am describing our extraction protocol below:
We started by preincubating samples in 250 無 lysosyme buffer for 30 min at 37蚓. After incubation Fermentas ThermoScientific Kit DNA extraction protocol was followed. 180 無 digestion buffer then, 20 無 Proteionase K were added, homogenized and incubated for 30 min at 56 蚓. 20 無 RNAz solution was added and incubated for 10 min. 400 無 50% ethanol was added and supernatant was transferred to column. Following washing procedures elusion buffer was added in order to izolate DNA.

What else should i do to increase DNA concentration?
cngl 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 09:59 PM.


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