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
- g025 T/B
- g031 T/B
- g034 T/B
- 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)
- pSPgRNA / g025
- pSPgRNA / g031
- pSPgRNA / g034
- 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
|
Haynes Lab - Short Projects
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