RNA agarose gel electrophoresis came up empty - (Jul/08/2014 )
What did the spectrophotometer curve look like? Did you get a 260:280 ratio? How about 260:230?
How long did you stain for? Did your ladder show up?
what is the condition of your formamide? it decomposes when in contact with water.
bob1 on Tue Jul 8 22:54:36 2014 said:
What did the spectrophotometer curve look like? Did you get a 260:280 ratio? How about 260:230?
How long did you stain for? Did your ladder show up?
For all my four samples, their respective data.
Sample 1: 546,56 ng/ul
A260/A280: 2.415
A260/A230: 0,770
Sample 2: 118,09 ng/ul
A260/A280: 2.101
A260/A230: 0,368
We add the Ethidium bromide when the gel is cooling but not solid. I haven't included ladder because we are short stock of it, due to holidays and delay in delivery. But everying is working in DNA electrophoresis.
Sample 3: 834,89 ng/ul
A260/A280: 2.666
A260/A230: 0,952
Sample 4: 317,06 ng/ul
A2360/A280: 2.237
A260/A230: 0,657
Thank you.
mdfenko on Wed Jul 9 12:53:38 2014 said:
what is the condition of your formamide? it decomposes when in contact with water.
It is not my prep, should I prepare one new?
Do you have any tips?
Thank you.
Thyago Leal on Wed Jul 9 13:36:40 2014 said:
mdfenko on Wed Jul 9 12:53:38 2014 said:
what is the condition of your formamide? it decomposes when in contact with water.
It is not my prep, should I prepare one new?
Do you have any tips?
Thank you.
you can deionize the formamide. here is the procedure used by beckman-coulter:
1) put 100ml formamide into a 250ml erlenmeyer flask. check the pH with pH paper (not electrode).
2) add 5gm mixed bed ion exchanger (ag501-x8(D) from bio-rad (catalog# 143-6425) or equivalent). stir with magnetic stir bar. if initial pH of formamide is less than 7 then stir for 45 minutes. if the pH is greater than 7 then stir for 60 minutes. keep the flask covered while stirring.
3) check the pH. if the pH is greater than 7 then continue. if the pH is less than 7 then discard it and start again with a fresh bottle of formamide.
4) filter through a 0.2um nylon filtration unit (nalgene #153-0020 or 151-5020 or corning #430771 or 430773).
5) store in single use aliquots at -20C in a non-frost-free freezer.
If your gel box is used to run classical plasmid mini-preps, it is grossly contaminated with RNAse
Your 260/280 ratios are odd. Please post a spectrophotogram for the members to see.
please post the photo of the gel and how you prepare the gel
For what it's worth, I regularly run non-denaturing RNA gels (w/o formamide/formaladehyde/urea) and see bands. so even if it were to decompose I'd still expect to see a band.