Haynes Lab:Notebook/Engineering PC-TFs/2014/03/22

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Summary

  • Miniprep on ligated plasmid CMV MV9 liquid culture.
  • Gel purification from prior CMV miniprep.

Performed mini-prep on each of the four picked colonies.

  • Ran concentration check on each:
Plasmid OD260 OD260/280 ng/μL
1. Colony 1 0.005 1.211 4.895
2. Colony 2 0.003 1.273 2.963
3. Colony 3 0.034 1.888 33.572
4. Colony 4 0.007 -5.308 7.215



Digest CMV


Restriction Digest Table

  • Checked plasmid minipreps with EcoRI/PstI digests
Reagent Volume Expected:
1. CMV = 588bp
DNA(CMV) 25.0 μL
10X buffer 3.0 μL
EcoRI 1.0 μL
PstI 1.0 μL
dH2O 0 μL
  15 μL --> 37°C/ 15 min.



Gel Purification: CMV/V0120

  • Using Prior Miniprep Digests
  1. CMV culture 1 - check with E/P
  • Used previously digested CMV.
  • Placed 15μL 1kB Ladder in well one and 30μL of CMV digest in well two.
  • Ran gel at 110V for 30 minutes.
  • Placed gel on UV transilluminator and cut out desired bands.
Hover name
15 μL/lane; 1% agarose; cut the brightest band furthest down at ~ 588bp; Ladder
  • Used Zymo gel purification kit.
  • Assumed 200mg gel, added 600μL ADB to each 1.5mL tube.
  • Put on heat block at 55°C for 5 minutes, vortexed and then placed back on the heat block for an additional 5 minutes, vortexed again.
  • Put ~750μL gel/ADB into spin column, centrifuged at max rpm for 30 seconds.
  • Pipetted 200μL wash buffer to each tube, centrifuged at max rpm for 30 seconds. Repeated once.
  • Transferred columns to new labeled 1.5mL tube.
  • Pipetted 15μL elution buffer to each column.
  • Spun at max rpm for 30 seconds.
  • Put marked (CMV gel pur2 3/22) CMV in -20°C fridge in box labeled Cameron.



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