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



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