I hate it when that happens! For the sample to fall to the bottom of the well it as to be denser then the surrounding buffer. Nathans right that loading dyes typically have either glycerol or sucrose to increase the density, and yes ethanol contamination by decreasing the density can cause samples to float away. Does Roche supply the composition of their buffer? It could be that an organic would decrease density enough to not allow it to enter the gel but this seems like a pretty big oversight on the part of Roche.
On Wednesday, March 20, 2013 3:37:15 PM UTC-4, phillyj wrote:
I did a restriction enzyme cut of a pcr product and wanted to run the--
gel and purify it. After adding the loading dye, I proceeded to put
the mix into the well. Unlike other times that I loaded, the mix just
seemed to float away. Only by being extremely careful and extremely
slow was I able to load a good amount. Usually I load 25uL in the well
but this time, I had to load all 45 uL and I think most of it didn't
get in the well.
The last time this happened, I was told it was ethanol contamination
from purification so I took utmost care to reduce this. I was doing
double digests so I did PCR clean up after the first cut. The only
thing different with this particular cutting is that I used Roche NheI
and SuRE cut Buffer M. The NheI had an odor to it; an organic smell
like DMSO. I have not had problems with NEB RE.
Any ideas?
Thanks
-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diybio@googlegroups.com. To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To unsubscribe from this group and stop receiving emails from it, send an email to diybio+unsubscribe@googlegroups.com.
To post to this group, send email to diybio@googlegroups.com.
Visit this group at http://groups.google.com/group/diybio?hl=en.
To view this discussion on the web visit https://groups.google.com/d/msg/diybio/-/VmBJDwHPVVcJ.
For more options, visit https://groups.google.com/groups/opt_out.
0 comments:
Post a Comment