Haynes Lab:Notebook/Short Projects/2015/05/28

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Ben Nyer - 28/05/2015

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

  • Follow the "Oligo annealing and cloning into backbone vectors - NEW" protocol

Phosphorylate and anneal oligo pairs

  1. g025 T/B
  2. g031 T/B
  3. g034 T/B
  4. g048 T/B
Reagent Rxn Mix (7x)
100 μM Oligo 1 1.0 ---
100 μM Oligo 2 1.0 ---
10x T4 Lign buf (NEB) 1.0 7.0
T4 PNK (NEB) 0.5 3.5
dH2O 6.5 45.5
  10.0


LabNet OptiMax Thermocycler: AnOlig RD

  • 37°C, 30 min
  • 95°C, 5 min
  • Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min]
  • 25°C, ∞


Dilute the product(s) 1:250

  • Add 2 μL product to 498 μL dH2O


Digestion/ Ligation Reactions (Vector / dsOligo Insert)

  1. pSPgRNA / g025
  2. pSPgRNA / g031
  3. pSPgRNA / g034
  4. pSPgRNA / g048
Reagent Rxn Mix (x7)
100 ng Vector 0.5 3.5
1:250 dsOligo 2.0 ---
10x FD buf 2.0 14.0
10 mM DTT 1.0 7.0
10 mM ATP 1.0 7.0
FastDigest BbsI 1.0 7.0
T4 DNA ligase 0.5 3.5
dH2O 12.0 84.0
  20.0

LabNet OptiMax Thermocycler: BbsI Dig/Lig

  • 6x [37°C, 5 min; 23°C, 5 min]
  • 4°C, ∞


Transformation(s)

  • Split ligations in 1/2, do rapid protocol for 1 set, long protocol for other set if needed
  • 10.0 μL ligation + 50 μL DH5α-turbo
  • Plate on 100 μg/mL amp


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