Cloning of guideRNA-Oligo into pLentiCRISPRv2: Difference between revisions
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{{Protokoll-Mix|titel=Materials|Inhalt=gRNA Oligos (sense/antisense; designed using ChopChop)<br>Vector pBluescript_U6-MCS digested with BbsI (Plasmid #3468)<br>T4 DNA ligase; 10x Ligase buffer<br>E. coli NovaBlue}} | {{Protokoll-Mix|titel=Materials|Inhalt=gRNA Oligos (sense/antisense; designed using ChopChop)<br>Vector pBluescript_U6-MCS digested with BbsI (Plasmid #3468)<br>T4 DNA ligase; 10x Ligase buffer<br>E. coli NovaBlue}} | ||
== Procedure == | |||
'''gRNA Oligo 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: | |||
{| class="wikitable" | |||
|95°C | |||
|4 minutes | |||
|- | |||
| colspan="2" |Start loop, 54x, 94°C, 1 min (-1°C/loop) | |||
|- | |||
|8°C | |||
|Store forever | |||
|} | |||
Revision as of 15:34, 30 May 2025
Materials
gRNA Oligos (sense/antisense; designed using ChopChop)
Vector pBluescript_U6-MCS digested with BbsI (Plasmid #3468)
T4 DNA ligase; 10x Ligase buffer
E. coli NovaBlue
Vector pBluescript_U6-MCS digested with BbsI (Plasmid #3468)
T4 DNA ligase; 10x Ligase buffer
E. coli NovaBlue
Procedure
gRNA Oligo 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:
| 95°C | 4 minutes |
| Start loop, 54x, 94°C, 1 min (-1°C/loop) | |
| 8°C | Store forever |