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 (5x)
100 μM Oligo 1 1.0 ---
100 μM Oligo 2 1.0 ---
10x T4 Lign buf (NEB) 1.0 5.0
T4 PNK (NEB) 0.5 2.5
dH2O 6.5 32.5
  10.0


Bio-Rad C1000 Touch Thermocycler: Olig PNK-Ann

  • 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 (x5)
100 ng Vector 0.5 2.5
1:250 dsOligo 2.0 ---
10x FD buf 2.0 10.0
10 mM DTT 1.0 5.0
10 mM ATP 1.0 5.0
FastDigest BbsI 1.0 5.0
T4 DNA ligase 0.5 2.5
dH2O 12.0 60.0
  20.0

Bio-Rad C1000 Touch Thermocycler: BbsI DigLig

  • 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