Haynes Lab:Notebook/Short Projects/2015/06/09

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Ben Nyer - 06/06/15

Cloning gRNAs into pSPgRNA for dCas9-HT binding to luc gene

Both cultures grown in 5 mL LB-Amp overnight, then purified using GenElute plasmid miniprep kit.


Nucleic acid quantification:

Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280
g031 1 589.70 10.2 1.9
g031 2 542.75 9.1 1.9

Restriction digest:

Sample ID Conc. (ng/µL) Vol. for 600ng (µL) Vol. water (µL)
g031 1 589.70 1.0 25.0
g031 2 542.75 1.1 24.9
Reagent Volume (µL)
DNA + water 26
10x FD Buffer (Green) 3
ApaLI 1
Total 30

Incubate at 37°C for 10 minutes, then run total volume on the gel. Expected size fragments are 1.2kb and 2.0kb.


Digest Results:

2015-06-09 g031 pSPgRNA ApaLI digestion annotated.png


Sequencing

Submit 200ng DNA + 1 µL of 10 µM primer (René's P139 primer works) in 10 µL total volume (make up difference with DNase-free H2O).

  1. g031-1
  2. g031-2