Re-use unused denatured/diluted Phix?
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bruce
May 19, 2009, 5:33:15 PM5/19/09
to solexa
If you follow Illumina's protocol for denaturing/diluting libraries
and use 1 lane for Phix then there is close to 900ul of denatured/
diluted Phix leftover after clustering with 120ul. Can the remaining
be stored and used effectively for future runs?? Illumina says no, but
guessing they want us to keep buying their very expensive phix.
Maybe it needs to be denatured again by heating/flash cool?
and use 1 lane for Phix then there is close to 900ul of denatured/
diluted Phix leftover after clustering with 120ul. Can the remaining
be stored and used effectively for future runs?? Illumina says no, but
guessing they want us to keep buying their very expensive phix.
Maybe it needs to be denatured again by heating/flash cool?
Scott J. Coutts
May 19, 2009, 6:09:19 PM5/19/09
to sol...@googlegroups.com
I think you probably could, but I've never tried it. I expect the
effective concentration would drop as some of the DNA adsorbs to the
sides of the tube. I guess there's only one way to find out :-)
Scott.
bruce
May 19, 2009, 7:27:39 PM5/19/09
to solexa
I did try it once a long time ago..... the effective concentration did
drop considerably, by about 60-70%. I didn't try again b/c this drop
concerned me at a time when I was having many other problems with the
platform. I'm ready to revisit this again to save a meager $18/run. I
thought the cluster #s dropped likely due to re-naturation of the phix
library fragments after freezing and thawing. How much of an effect
does adsorption to the tube have? Doesn't Tween counter this effect?
If re-naturation is an issue as well, I assume 95C and snap cool would
denature again (probably wouldn't be good to denature with NaOH again)
On May 19, 6:09 pm, "Scott J. Coutts" <Scott.Cou...@med.monash.edu.au>
wrote:
drop considerably, by about 60-70%. I didn't try again b/c this drop
concerned me at a time when I was having many other problems with the
platform. I'm ready to revisit this again to save a meager $18/run. I
thought the cluster #s dropped likely due to re-naturation of the phix
library fragments after freezing and thawing. How much of an effect
does adsorption to the tube have? Doesn't Tween counter this effect?
If re-naturation is an issue as well, I assume 95C and snap cool would
denature again (probably wouldn't be good to denature with NaOH again)
On May 19, 6:09 pm, "Scott J. Coutts" <Scott.Cou...@med.monash.edu.au>
wrote:
ClusterJunkie
Jun 16, 2009, 11:33:03 AM6/16/09
to solexa
Hi Bruce-
We have had pretty good succes at Rosetta with re-using the material
stored in hyb solution. This includes using our actual libraries as
well. While modest drops in cluster density can occur with time, we
have not witnessed the large percentage drop that you mentioned in
your post above. One thing we are careful to do is very thoroughly
vortex the sample prior to transfer to strip tubes. For reference, we
use Axygen microfuge tubes. It is indeed possible that plastics from
different vendors can hang onto dilute DNAs in differing degrees,
though I don't expect that this is a major problem given that there is
detergent in the hybridization buffer.
Matt
We have had pretty good succes at Rosetta with re-using the material
stored in hyb solution. This includes using our actual libraries as
well. While modest drops in cluster density can occur with time, we
have not witnessed the large percentage drop that you mentioned in
your post above. One thing we are careful to do is very thoroughly
vortex the sample prior to transfer to strip tubes. For reference, we
use Axygen microfuge tubes. It is indeed possible that plastics from
different vendors can hang onto dilute DNAs in differing degrees,
though I don't expect that this is a major problem given that there is
detergent in the hybridization buffer.
Matt
Johar Ali
Jun 16, 2009, 11:45:32 AM6/16/09
to sol...@googlegroups.com
Is it worth running a lane of Phix?
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