Cloning of shRNA into lentiviral vector pLKO.1: Difference between revisions

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Created page with "== Procedure == === Primer annealing === * annealing reaction contains: 1 µl sense primer (stock solution 100 µM), 1 µl antisense primer (stock solution 100 µM), 5 µl NEB buffer 2 and 43 µl ddH<sub>2</sub>O * Annealing reaction occurs in thermocycler with the corresponding program:"
 
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* annealing reaction contains: 1 µl sense primer (stock solution 100 µM), 1 µl antisense primer (stock solution 100 µM), 5 µl NEB buffer 2 and 43 µl ddH<sub>2</sub>O
* annealing reaction contains: 1 µl sense primer (stock solution 100 µM), 1 µl antisense primer (stock solution 100 µM), 5 µl NEB buffer 2 and 43 µl ddH<sub>2</sub>O
* Annealing reaction occurs in thermocycler with the corresponding program:
* Annealing reaction occurs in thermocycler with the corresponding program:
{| class="wikitable"
|+
|1
|Hot start
|'''94°C'''
|Pause – addition of enzyme mix
|-
| colspan="4" |''<small>7 repetitions of steps 2-4 with hybridization temperature - 1 ºC per cycle (touch-down)</small>''
|-
|2
|Denauturation
|'''94°C'''
|20 seconds
|-
|3
|Hybridization
|'''64-58°C'''
|20 seconds
|-
|4
|Elongation
|'''72°C'''
|depending on fragment size (1 min/750 bp)
|-
| colspan="4" |''<small>30 repetitions of steps 2-4 with hybridization temperature of 58 ºC</small>''
|-
|5
|Elongation
|'''72°C'''
|5-10 minutes
|-
|6
|Stop
|'''8°C'''
|
|}

Revision as of 12:14, 30 May 2025

Procedure

Primer annealing

  • annealing reaction contains: 1 µl sense primer (stock solution 100 µM), 1 µl antisense primer (stock solution 100 µM), 5 µl NEB buffer 2 and 43 µl ddH2O
  • Annealing reaction occurs in thermocycler with the corresponding program:
1 Hot start 94°C Pause – addition of enzyme mix
7 repetitions of steps 2-4 with hybridization temperature - 1 ºC per cycle (touch-down)
2 Denauturation 94°C 20 seconds
3 Hybridization 64-58°C 20 seconds
4 Elongation 72°C depending on fragment size (1 min/750 bp)
30 repetitions of steps 2-4 with hybridization temperature of 58 ºC
5 Elongation 72°C 5-10 minutes
6 Stop 8°C
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