User:Christine Doan/Notebook/Identifying Kinase Substrates of PfCK2/2014/02/24: Difference between revisions
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** Save 25ml for induction gel | ** Save 25ml for induction gel | ||
** Use remaining 225ml according to that aliquot | ** 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== | ==Sub 4== |
Revision as of 13:19, 27 February 2014
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CK2
1. Transform
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
Sub 4
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