User:Christine Doan/Notebook/Identifying Kinase Substrates of PfCK2/2014/02/24

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CK2 New CK2 was successfully sequenced Don't trust possible discrepancies between 50ml induction Western and 250ml samples in -80C Re-transform new CK2 from stock into BL21RIL Grow up 250ml to OD Save 25ml for induction gel Use remaining 225ml according to that aliquot Proposed protocol 1. Transform Stock #1356 (1/29/14 pGEX-4T-3 CK2α from Christian Doerig) BL21-CodonPlus-RIL Incubate Amp plate ON 2. Select colonies, grow in 5ml LB + Amp ON 3. Inoculate 2.5ml into 250ml LB, grow to OD600 = 0.5-0.6 4. Induce with 0.5mM IPTG, incubate 20hrs at 20C 25ml: spin, wash, spin, resuspend, add lysozyme, lyse, spin, save pel and sup Run two sides of one gel: stain one, Western one 225ml: spin, resuspend in MK buffer + protease inhibitor, save in -80C Sub 4 Transform Andi's Sub 4-2 (protein kinase, putative) into BL21RIL also Check pET-30 info for induction conditions