Experiments This Week
Monday – Third 20% SDS-PAGE gel of the oligos – 1st on Typhoon instrument; failed; one band seen; lots of background. Need to rinse gel to get rid of background fluorescence. Made solutions of rtcB varying NaCl and DTT.
Tuesday – two 12% rtcB SDS-PAGE gels, of different buffers. Varied NaCl, DTT and pH. None of the protein ran.
Wednesday – planning and buffer prep for prot re-expression; organized rtcB articles and purification protocols; typhoon account setup – user ID: Salaita Lab; password: salaitalab533
Thursday – expressing of Keio rtcA::km/pQE70_rtcB::amp (2nd time); froze pellets at -80C
Friday – making up buffers for next week (according to Shuman’s protocol)
Purchases for the week = $62.28
Glacial acetic acid (refill stock), $18.21, 5-22-13
Methanol, Histological grad (refill stock), $12.60, 5-22-13
500 ml wash bottle, $7.54, 5-22-13
Sucrose, omnipure; $23.93, 5-24-13
Tentative Schedule for Next Week
Monday – Finish making buffers; protein purification
Tuesday – gel of protein purification
Wednesday – planning day/inoculation of culture
Thursday – if expression doesn’t work; re-express protein
Friday – re-purify protein
Tips
Rinse the gel stained with SYBR gold to avoid background fluorescence
Filter sterilizers are really expensive – about $300 for a box of 100 of the 1 ml sterilizers