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RNA Gel 3 Sharp bands?

RNA 3 bands

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#1 Rich2788

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Posted 31 October 2014 - 09:26 AM

Hi all, 

 

I did a RNA extraction today on C.reinhardtii (green algae). I used the Trizol PURELINK kit from Ambion, with a few alterations suggested in this thread. I used Qiagen DNase on column. 

 

I used a Nanodrop to check the absorpsion. I got a 260/280 ratio of 2.13-2.18 and a 260/230 ratio of 2.4-2.41. Higher than  values I have measured before I used the tips to improve yeild and lower 230 contamination. 

 

I ran a non-denaturing gel (1% agrose TEA buffer) and got three sharp bands at 1500 bp, 1000 bp and 750 bp. I've previously got less sharp bands at 1500 and 750 )(assuming 25S and 18S). What is this third band? I ran an Image J analysis and the ratio of the band intensity is about 1:1:1 compared to 1.5:1 I have been getting previously. My PI is stumped. Any ideas please?

 

The gel image attached (31-10) and an image of a previous extraction. Hopefully you can see them. 

 

 

Attached Thumbnails

  • 31-10 RNA 3 bands.jpg
  • Before method alterations - two band.jpg


#2 bob1

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Posted 31 October 2014 - 11:15 PM

It could be one of the other rRNA bands, but normally you would also see a mRNA smear in the lower part of the gel if one of these was to be obvious.






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