Week of June 14th Summary
Experiments This Week
Monday – 2nd 12% SDS-PAGE rtcB gel, to confirm it’s not aggregating. It ran ok, except for the 1x PBS, which had aggregated. The -20C stocks are still good. Setup a ligation reaction (4th) o/n, in water bath, in duplicate.
Tuesday – 20% SDS-PAGE of the ligation reaction and a gel of Kevin’s old FAM substrate. RNasin enzyme that I ordered came today.
Wednesday – attempted to pour and store four 20% gels, but three of them failed. Stored the 4th in the cold room.
Thursday – My new RNA substrates arrived today. Read about RNA gels. Bought Rainex, for the short gel plate. Did more ligation planning.
Friday – Made up my RNA solutions for the ligation reaction today. Nanodropped the RNA and made stocks. Setup a ligation reaction overnight (5th).
Saturday – Poured an RNA 20% gel, but it failed. Need to mix the TEMED before adding APS, since the 15 ml falcon tube I was using is narrow and it doesn’t mix well. Wells did not form. Leaked from the top. Ran the reaction on the stored 20% gel and imaged, but it looked bad.
Purchases for the week = $69.08
rtcB in 1x PBS lasts about 11-13 days in the cold room (4C). It precipitated out after that.
When using the glycine buffer, protein gels run completely in 30 min, not 35 min! However, even though the dye will bleed out the bottom, the protein itself will still be higher up. I think 35 min is ok to run it in.
Daniel recommended using barrier tips, even with the hybrid sequences. Also, wipe off my pipets with H2O2.
On IDT, 100 nmole is the amt of starting material. You don’t necessary get back that much. I’m using .1 nmole in each experiment, so I can still do 70 experiments with 7 nmole – which is about what they guarantee to send.
I really need a ssDNA or RNA ladder, to really tell what is going on with my substrates.
Make sure to mix the TEMED before adding APS, if doing the reaction in a narrow falcon tube.