LaCava Research Wiki

AIM/HYPOTHESIS

Given the results of 2. Testing Ribonucleoside Vanadyl Complex vs. RNasin_Dec 20 2019 we want to test the hypothesis that RVC needs to be depleted prior to running samples on the Bioanalyzer, because its presence might interfere with the fluorescent readout. Hua has tried RVC on MT302 before (09/09/19 - RNVC Testing – RNA Prep from MT302 and 162T) and there it looked solid, so we should be able to repeat that.

Also, the samples that were run later have peaks at only halve the length compared to the peaks in the first run.

MATERIALS

• Nuclease free extraction buffer (20mM HEPES, pH7.4, 1% TritonX-100, 500mM NaCl, 1x protease inhibitor); RNAse inhibitor at following concentrations.
• Scale: 50mg cell powder per IP reaction with 10ul of beads

1. MT302 anti-ORF1 1:250 RNasin EB; 1:1000 RNasin wash (positive control)
2. MT302 anti-ORF1 no RNAse inhibitor (negative control)
3. MT302 anti-ORF1 Ribonucleoside Vanadyl Complex 10 mM EB+wash
4. MT302 anti-ORF1 Ribonucleoside Vanadyl Complex 10 mM EB+wash; depletion of RVC by adding 100mM EDTA . 

METHODS

Vanadyl was heated at 65°C before use, resulting in a homogenous black/green solution as stated by the manufacturer.

RESULTS

DISCUSSION

Notes;

• The beads in vanadyl supplemented buffer did not stick to the magnet as they normally do. Somehow the VRC might interfere with this magnetic interaction. For now we will just be careful during the wash steps.
• Furthermore, I added only 15 µL of 0.5M EDTA (pH 8.0) to the elution (total of 225 µL, so 33mM), while this should have been more.
• Finally, some samples contained much more liquid after RNA elution than 6 µL.

In the end, I decided not to run these samples but to retry the experiment, and then run the samples together.