I used the columns that you can fill yourself with the HPLC machine. Don't know how much does it cost though. For DNA and some bigger stuff, I think I used Sepharose and Agilent used to have the whole range that you can choose from depending on what resolution you need. Regarding the dye labeling, I actually meant the covalently modified DNA. I used that before and of course that is stable; I admit that would be expensive though. Typically people run stuff sequentially and compare times but there is a variation from run to run, so it is more reliable and faster if you can do it in just one run.
-- Huge pressure is a bummer for HPLC. If we could do it in gas phase, it would be lower pressure for sure, less adsorption to the column material too and possibly faster (that would reduce the resolution though). I know that they gas chromatography is used for volatile stuff but I am puzzled why it could make it work for lyophilized stuff for example.
On Tuesday, March 3, 2015 at 11:32:04 PM UTC-6, Nathan McCorkle wrote:
Where did you get HPLC machine? They look pretty expensive on Ebay.
On Tuesday, March 3, 2015 at 11:32:04 PM UTC-6, Nathan McCorkle wrote:
I just did HPLC today on caffeine and it was pretty easy... I was
checking column prices last week on ebay and they seemed like around
$50 for used... but what about new?
What are the ideal DNA columns? Is there an easy/cheaper DIY
compromise/substitute available? (maybe silica?)
On Wed, Feb 18, 2015 at 11:57 AM, GO <gobi...@gmail.com> wrote:
> We can also co-pass a already known strands with some fluoro or other
> label that can be detected at wavelength other than 260 nm (this would serve
> as a ladder in electrophoresis).
Concern might be that the dye dislocates during chromatography...
> Also its more difficult to collect DNA
> later in gel compared to HPLC. Liquid chromatography can be used to detect
> other things as well (not just charged DNA) so it's even more useful for DIY
> people.
I didn't collect elutions, but I think I would have just had to figure
out the lag time between the detector and the end of the waste tube.
It certainly would have been easy. The one problem I could see is that
while the sample loop was only 100uL, it was recommended to flush with
1000uL (to clean out any trace of previous sample) and I was using
more like 2 or 3 mL since we had plenty. So for people with only 50uL
or even 500uL, we need to figure something out... as a gel can be
loaded with something like 10-20uL. I am not sure if diluting it to
1000uL would be smart or not... I am sure the required volume could be
decreased, but there is still the flush volume to prevent
cross-contamination... I guess this could be switched from sample
being used to wash/rinse to DNA buffer.
I think the high-pressure generation and injector valve is the hard
part of this. Maybe a good reason to go after capillary (or capillary
gel electrophoresis) electrophoresis instead... no pressure to worry
about, so adding sample shouldn't be much different for end-users used
to loading gel wells.
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