Hi all,
I am in the midst of extracting my snail tissue samples via Omega BioTek's EZNA Mollusc specific DNA extraction kit and I am having some shearing/degradation.
I extracted two samples as a test run and they produced a nice, clean band but every sample after I have gotten varying levels of shearing. I have been informed that the DNA is fine for downstream application but this is more for my own sanity...I really don't see what I am doing differently between the extractions. Have I introduced a small amount of DNase contamination? There are vigorous vortexing steps necessary to the removing of contaminants but I did those for all extractions I have done, including the first two.
All the best!
Brenna
Hi Brenna,
I totally agree wit Artur. One thing I would say, try not to vigorously vortex for a long period of time(45 seconds is enough). The very long period vortex during extraction procedure would degrade your DNA.
Thank you to both of you!
To Artur, I loaded the same sample volume but after Qubit-ing my samples I did load quite a bit more DNA the second gel. Tweaked my extraction protocol and it seems to be working for me to get more concentrated DNA. I loaded the same amount of ladder (7 uL) both times and ran it for an extra five minutes at 100V in the second gel. I did switch pipettes between gels and they are in desperate need of calibration. I will play around and see if I can't get a clearer gel like the first time. Thank you for the input and putting my mind at ease with their quality.
To Fawaz, my protocol has a maximum of 15 second vortexes, which are done twice and necessary to removing inhibitors and precipitating the DNA. I try to minimize freeze/thaws with my samples. Thank you for the input as well!
Cheers,
Brenna
Hi Brenna,
I guess you have done everything right....
have you changed the buffer ????
you can use a low voltage as well....
If there is no issue, then there might be an issue with the gel doc machine.
Like if you place a gel in the gel doc, its wet, it might slip while the gel doc is taking picture.
so due to low light and a slow shutter speed of the camera, the pic will be like the one you have uploaded. But i dont think this is an issue.
Best of luck.
Thank you to both of you! Sorry for the delay in my response...lab work got put down for a few days as I've been getting in the groove of TAing this spring.
I think our running buffer is at the end of its life (been re-used close to 15X) and that our pipette is actually pipetting more than 7uL. The wells should be able to handle up to 10uL and I have run that ladder at 7uL previously and hadn't had the same issues.
Pipettes are being calibrated and I will make fresh running buffer. I am not too worried since I think my DNA is actually fine and at in end of the day that's what matters.
Thanks everyone!
Brenna
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