Haynes Lab:Notebook/Engineering PC-TFs/2014/04/18

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Summary

4/16

  • Cloned mv9cmv construct into DH5a-T cells using standard procedure. Incubated overnight.

4/17

  • Removed plates from incubator.
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;
  • Plate 1 is the experimental plate with growth. Plate 2 is the negative control and had no growth (debris is visible underneath the plate).

4/18

  • Grew up liquid cultures from prior night.
  • Miniprepped cultures.
Plasmid OD260 OD260/280 ng/μL
1. Colony 1 0.202 1.905 162
2. Colony 2 0.253 1.92 203
  • Restriction Digest
Reagent Volume Expected:
1. CMV = 643bp
DNA(CMV/MV9) 4.0 μL
10X buffer 1.5 μL
NotI 1.0 μL
NruI 1.0 μL
dH2O 7.5 μL
  15 μL --> 37°C/ 15 min.
  • Verified for the sake of not wasting money on sequencing in the case that something unexpected occurred.
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15 μL/lane; 1% agarose; 
Ladder
  • Expected base pair length: 643
  • Observed base pair length: ~650



  • Sent off for sequencing
Plasmid Title Primer Water Plasmid (~200ng/uL)
1. cmvmv9f1 FP 1uL DD122 B 8uL 1uL
2. cmvmv9r1 RP 1uL DD123 B 8uL 1uL
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