User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/19

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Karmella's BioBrick Cloning Main project page
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05/19/15

  • Cas-tone - cultures, minipreps, digests
  • Rene - PCR library 96-well plate



Cas-tone - cultures, minipreps, digests

  • Minipreps
    • Check with FD E/P digests
    • Modification (dsOligo insert replacing VP64) removes an EcoRI site
Reagent Volume Expected:
1,2. CMV-dCas9-HA = 5317, 4189
CMV-dCas9-VP64 =5459, 3016, 1337
Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~10 min.

CONCLUSIONS

  • Success! Keep clone #1 (lane 1). Continue to next step: histone insertions



Rene - PCR libraries

  • Inoculate 200 μL mini-cultures with individual colonies picked from PCR cloning plates with sterile micropipette tips
  1. Luc14g034: 192 colonies, 2 96-well plates (A and B)
  2. Gal4EEDg034: 192 colonies, 2 96-well plates (A and B)
  • Seal with parafilm, grow at 37°C with shaking
  • Spot cultures from 96-well plates onto LB agar amp (100μg/mL) using multichannel pipettor
    • Transferred 2 μL from each well to a 6x8 "array"
    • Each 96-well set was spotted onto two plates (A1-G1, A7-G7)
    • 8 plates total



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