RNA electrophoresis
Preparation of the samples:
Work RNase-free! Treat your workspace with RNaseZAP prior to your experiment and work with gloves. Don't talk while handling your samples!
- Prepare the electrophoresis samples in 0.2 mL PCR tubes; 2 µL of 2X RNA Loading Buffer per 2 µL of sample (check the appropriate preparation for each marker).
- Put your samples at 90 °C for 5 minutes.
- Leave your samples on ice for 2 minutes. Store your samples at 4 °C when not loaded directly on the gel
Preparation of the gel:
Work clean! Handle all material labelled as EtBr contaminated with gloves. Don't take it outside of the EtBr area and don't touch anything that is not labelled as EtBr contaminated with gloves.
- Prepare TBE buffer: take the 10X concentrated TBE from the chemicals cabinet and dilute it 10 times with milli-Q.
- Weigh agarose for a 2% gel. For 100 mL, 2 g of agarose is necessary.
- Mix the TBE solution with the agarose and heat the solution (in a microwave) until it is completely dissolved.
- Add 10,000 X SYBR Safe to the solution and mix well. Meaning that for a large gel of 100 mL you have to add 10 µL of SYBR Safe.
- Pour the solution into the mould, making sure there are no bubbles. Add a comb to create wells for the samples. Let it solidify (~10 minutes).
RNA electrophoresis:
- Transfer the gel to the electrophoresis cell minding the arrow that indicates the direction of DNA/RNA migration. Remove the combs and cover it with TBE-buffer.
- Load the RNA ladder in the first well (check the appropriate volume for each marker) and load 4 µL of the samples in the other wells, according to the order in your lab journal.
- Connect the cables following the colour code and run at 80 V for 60 min.