Haynes Lab:Notebook/Short Projects/2015/06/02

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Ben Nyer - 02/06/15

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

All 7 cultures showed growth in LB-Amp liquid medium. Performed plasmid minipreps and eluted in 50 µL DNase-free H2O.


Nucleic acid quantification:

Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280
g025 1 756.7 18.69 1.93
g025 2 670.6 13.37 1.93
g031 1 558.2 10.53 1.93
g034 1 617.4 8.81 1.92
g034 2 570.4 16.40 1.93
g048 1 660.7 9.80 1.92
g048 2 565.5 6.63 1.91

Restriction digest:

Part Conc. (ng/µL) Vol. for 600ng (µL) Vol. water (µL)
g025 1 756.7 0.8 25.2
g025 2 670.6 0.9 25.1
g031 1 558.2 1.1 24.9
g034 1 617.4 1.0 25.0
g034 2 570.4 1.1 24.9
g048 1 660.7 0.9 25.1
g048 2 565.5 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

2015-06-02 gRNA pSPgRNA ApaLI digestion annotated.png

shadow effect probably because the table was bumped during imaging.


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. g025-1
  2. g025-2
  3. g031-1
  4. g034-1
  5. g034-1
  6. g034-2
  7. g048-1
  8. g048-2