This page has moved to here: http://protocol-place.com/basic-lab-techniques/rna-purification/rna-extraction-from-collagen-gels-using-qiagens-rneasy-kit/
Thanks for the protocol- what do your 260/230 ratios look like and what is your average yield? I’ve been following a similar protocol and am disappointed in my RNA yield. Just wondering how mine compares to yours! Thanks again.
Thanks for the comment. I have addressed your questions in the page called “Measuring RNA with NanoDrop” – see https://protocolplace.wordpress.com/basic-lab-techniques/rna-protocols/measuring-rna-yield-with-a-nanodrop-spectrophotometer/ . I just updated this protocol so that it links to that page now (Step 20).
You didn’t mention how many cells are in your samples, but I’ve noticed that samples with fewer cells yield less RNA and also lower A260/230 ratios. Try increasing cell numbers if you think that having too few cells might be an issue. It could be that your cells are dying too, causing less yield and lower quality. For example, if you are making cell-seeded collagen gels, be sure the pH of the collagen is neutral each time and if you can check any other way, be sure that your cells are actually surviving the experiment. See below for more info, and good luck!
Copied from the other page mentioned above:
“As a reference, collagen gels seeded with ~700,000 cells typically yield around 50-150 ng/µL of RNA when eluted in 30-35 µL of water.”
“The A260/A230 ratio should also be above 2.0. A low A260/230 ratio indicates contamination with the wash solutions, chaotropic salts, phenols or protein. A low A260/A230 ratio is most likely due to contamination of the samples with washing buffers during the Minispin tube washes. Be more careful when handling the tubes, especially when adding wash solution or removing the spin-through. Try to gently pour out the flow-through and then carefully wipe away drops on the outer rim of the collection tube with a KimWipe.”
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