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
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Volume
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Expected: 1,2. CMV-dCas9-HA = 5317, 4189 CMV-dCas9-VP64 =5459, 3016, 1337
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15 μL/lane; 1% agarose; Ladder
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DNA(plasmid) |
2.0 μL
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10X buffer |
1.5
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EcoRI |
1.0
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PstI |
1.0
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dH2O |
9.5
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15 μL
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--> 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
- Luc14g034: 192 colonies, 2 96-well plates (A and B)
- 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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