SEQanswers

SEQanswers (http://seqanswers.com/forums/index.php)
-   454 Pyrosequencing (http://seqanswers.com/forums/forumdisplay.php?f=5)
-   -   DNA Shearing for 454 (http://seqanswers.com/forums/showthread.php?t=766)

alanc 11-07-2008 01:08 AM

DNA Shearing for 454
 
I came across this technology for DNA shearing http://www.kbioscience.co.uk/instrum...ept%202008.pdf. Has anyone got any experience with this technology?

jOn 11-08-2008 05:10 AM

I saw this poster being presented at a recent GS-FLX userís group meeting in London. I got the impression that 454 Branford had primarily implemented this technology in order to scale up their sample throughput.

gilly 11-27-2008 01:13 AM

Using the Covaris for shearing DNA has both advantages and disadvantages.

On the plus side you can massively reduce the amount of input DNA, weíve made shotgun libraries using 500 ng of DNA. On the down side however itís not as reproducible as using nebulisation.

The attached are screenshots of Agilent traces (without SPRI bead clean up) where the following settings have been used:
Duty cycle = 20%
Intensity = 3
Cycle burst = 200
Time = 45 seconds.

Peak on 1 is at around 640 bp and about 100 bases lover on 2.

http://img256.imagevenue.com/loc68/t...1_122_68lo.jpg
http://img111.imagevenue.com/loc1197...122_1197lo.jpg

Both samples contain 5 ug of DNA in 100 ul of TE. The smaller sized tube was used (max capacity 130 ul) with the fiber in place. Itís also important to use the metal crimped cap as lots of the energy is lost using the plastic ones.

As things stand I wouldn't recommend switching all DNA shearing over to this system but it can be very useful when less starting material is available.

Anthony 02-26-2009 02:27 AM

The graphs seem to indicate that there may be a sample viscosity issue and you might want to try some of the following ideas,
-What is the mass of DNA, if it is more than 3ug it is worth trying a "pre-conditioning step"
Simply raise waterbath to 20degC and run a short "blast" 5 secs, (20% duty cycle, level 5 intensity, 200 cycles per burst) prior to the desired fragmentation dose. Up to 20ug/100ul have been run under these conditions.


All times are GMT -8. The time now is 03:57 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.