RNA Clean up

 We started the week with RNA isolation, then did a clean up kit followed by a gel and fluorometer analysis. We did the normal RNA isolation procedure, then after analyzing the results did a clean up the next day. We did a Midori green RNA gel (1%, 50ul gel with 5ul of Midori Green) from those results, and analyzed the sample quality the next day.


RNA:

1. Resuspend a fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bead bashing lysis tube

2. Secure the tube on a bead beater with a 2ml tube hold assembly and process. Repeat 5 cycles of 1 minute on and 2 minutes on ice

3. Centrifuge the tube for one minute to pellet debris

4. Transfer up to 400μl of the cleared supernatant into a Zymo-Spin IICG column in a collection tube and centrifuge for one minute. Retain the flow through

5. Add an equal volume of ethanol (95-100%) and mix thoroughly

6. Transfer the mixture into a Zymo-spin IICR column in a collection tube and centrifuge for one minute. Discard the flow through

7. Add 400μl RNA wash buffer to the column, centrifuge for one minute, and discard the flow through

8. Prepare the DNase 1 reaction mix (75μl DNase buffer and 5μl DNase per tube), add 80μl directly to the column matrix, and incubate at room temperature (20-30°C) for 15 minutes before continuing purification

9. Add 400μl RNA prep buffer to the column and centrifuge for one minute. Discard the flow through

10. Add 700μl RNA wash buffer to the column and centrifuge for one minute. Discard the flow through

11. Add 400μl RNA wash buffer to the column and centrifuge for one minute. Discard the flow through

12. Add 400μl RNA wash buffer to the column and centrifuge for two minutes. Discard the flow through

13. Transfer the column to a nuclease-free tube, add 50μl DNase/RNase-free water directly to the column matrix, and centrifuge



ng/ul

a260/280

260/230

A

35.32.010.53

B

85.21.951.54

C

133.0

1.670.60



Clean up:

1. Add 100ul RNA Cleanup Binding Buffer to the sample 

2. Add 150ul Ethanol to your sample and mix by pipetting or flicking the tube. Do not vortex.

3. Insert column into collection tube, load sample onto column and close the cap. Spin for 1 minute. Discard Flow through

4. Re-insert column into collection tube. Add 500ul RNA wash buffer and spin for 1 minute. Discard flow through.

5. Repeat wash. Add 500ul RNA wash buffer and spin for 1 minute. Discard flow through.

6. Transfer column to an RNase-free 1.5 ml microfuge tube

7. Elute in 50ul nuclease-free water


For the Fluorometer we did 3 samples and 3 standards.

We made the reagent with 7ul of dye and 1393ul of buffer, for 1400 total as we calculated with an extra tube just in case. 

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