User:Christine Doan/Notebook/Identifying Kinase Substrates of PfCK2/2014/02/26: Difference between revisions

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==Protein Kinase CK2 in Pf==
==CK2==
[[image: OldandnewCK2.png]]
* Andrew replated transformation
* Nothing at all
* Will have to retransform
* Mystery: DNA stock was a miniprep from colony grown from transformation made when I needed to sequence
** The original transformation plate worked, hence the plate growth and colony selection
** Secondary transformation did not work
** Will do 2 transformations: one from the "stock" and one from the miniprep that was used to make the "stock"


==Kinase Substrates==
==Sub 4 mini-inductions==
* Both colonies yielded liquid cultures
* Inoculated 1.5ml of A into 150ml LB + 150ul Kan in 500ml flask
* Grew to OD600 = 0.6 (~1.5hrs)
* Aliquoted 25ml into 6 125ml flasks
{| class="wikitable"
|-
! Volume in ul (for 125ml)!! IPTG (mM) !! Temp !! Time (hr) !! IPTG (mM) !! Temp !! Time (hr)
|-
| 10 || 0.4 || 37 || 2 || 0.4 || 25 || 6
|-
| 20 || 0.8 || 37 || 2 || 0.8 || 25 || 6
|-
| 25 || 1.0 || 37 || 2 || 1.0 || 25 || 6
|-
|}
* Save 1ml UI sample, spin, wash, resuspend, store -80C
* Cool flasks labeled 25C for 10mins before adding respective concentrations of IPTG
* At the end of all inductions:
** Save 1ml total I sample from each, spin, wash, resuspend, store -80C
** Rest: spin, wash, resuspend, add protease inhibitor, save 5ml I sample for lysing and observing protein solubility, store all -80C




Revision as of 10:01, 27 February 2014

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CK2

  • Andrew replated transformation
  • Nothing at all
  • Will have to retransform
  • Mystery: DNA stock was a miniprep from colony grown from transformation made when I needed to sequence
    • The original transformation plate worked, hence the plate growth and colony selection
    • Secondary transformation did not work
    • Will do 2 transformations: one from the "stock" and one from the miniprep that was used to make the "stock"

Sub 4 mini-inductions

  • Both colonies yielded liquid cultures
  • Inoculated 1.5ml of A into 150ml LB + 150ul Kan in 500ml flask
  • Grew to OD600 = 0.6 (~1.5hrs)
  • Aliquoted 25ml into 6 125ml flasks
Volume in ul (for 125ml) IPTG (mM) Temp Time (hr) IPTG (mM) Temp Time (hr)
10 0.4 37 2 0.4 25 6
20 0.8 37 2 0.8 25 6
25 1.0 37 2 1.0 25 6
  • Save 1ml UI sample, spin, wash, resuspend, store -80C
  • Cool flasks labeled 25C for 10mins before adding respective concentrations of IPTG
  • At the end of all inductions:
    • Save 1ml total I sample from each, spin, wash, resuspend, store -80C
    • Rest: spin, wash, resuspend, add protease inhibitor, save 5ml I sample for lysing and observing protein solubility, store all -80C



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