Monday 06/29/15

9:45 am – 5:15 pm

Testing RNAi knockdown

RNA isolation

(assuming you cultured in 6 well plates – scale accordingly)
* Wash cells in cold PBS (with Kc, might have to resuspend, spin down, wash in PBS, then spin down again)
* resuspend cell pellet in 350 ul of RLT Buffer (from QIagen RNeasy plus kit). Vortex for 30 seconds. I find that the sample is stable if stored at -80oC in this. You have to make sure you vortex before storage though.
* follow Qiagen manual for remaining steps to purify RNA, including the “gEliminator” columns to remove genomic DNA.

First strand cDNA synthesis

• protocol

RNA dilutions

• 5 ug of RNA (2.5 ug/uL) -> 2 uL RNA
• 5 uL ddH2O (for 2 uL starting RNA) (40 uL)
• 1 uL oligo-dT primer (9 uL)
• 1 uL dNTP mix (9 uL)
• heat to 65C for 5 min, return to ice.

per reaction (master)

• 2 uL 10x buffer (18 uL)
• 4 uL MgCl2 (36 uL)
• 2 uL DTT (18 uL)
• 1 uL RNase OUT (9 uL)
• all samples with RT (1 uL per reaction)
• all samples without RT

sample order

1. Pc + RT
2. Esc + RT
3. Pc, Psc, Esc, Scm + RT
4. water + RT
5. Pc no RT
6. Esc no RT
7. Pc, Psc, Esc, Scm no RT
8. water no RT

RNAi experiment progress

• Cells treated with RNAi, Tuesday evening 6/23
• Incubated 6/24-6/29. Fixed 6/29.