Cloning of guideRNA-Oligo into pLentiCRISPRv2 - Revision history

ESO wikiadmin at 14:35, 30 May 2025

2025-05-30T14:35:30Z

← Older revision		Revision as of 16:35, 30 May 2025
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	\|Store forever		\|Store forever
	\|}		\|}

			== Preparation of BsmBI digested vector pLentiCRISPRv2 ==

			* Digestion of about 4 µg pLentiCRISPRv2 with 2 µl BsmBI in a total volume of 50 µl over night at 37° C
			* Purification of vector via agarose-gel electrophoresis (expected size about 13000 bps) and Gel extraction kit. Elute in 50 µl EB buffer.
			* Check amount of purified, digested vector on agarose gel. Freeze down 1 µl aliquots for future ligations.

			== Ligation, Transformation and Analysis of Clones ==

			* Ligation of vector and annealed oligos: 1 µl digested pLEntiCRISPRv2 (about 100 ng), 1 µl annealed gRNA oligo pair, 1 µl T4 ligase, 1µl T4 ligase buffer and 6 µl ddH<sub>2</sub>O; incubation overnight at 4° C. (Neg. control: ligation reaction of vector only w/o annealed oligo pair)
			* Transformation in ''E.coli'' NovaBlue (plating on LB- Amp), Grow on 30°C ! the next day 10 clones are tested via colony PCR
			* Analysis via colony PCR forward primer (sense oligo of used gRNA pair), reverse primer #2708, product: 530bp
			* positive control: plasmid No. 4300 (pLentiCRISPRv2 sgCEACAM3.1), primers #2710 + #2708; PCR product of about 530 bps;
			* negative control: plasmid No. 3777, primers #2708 and sense oligo of used gRNA pair; no PCR product
			* analysis via 2% agarose gel
			* Sequencing of positive clone: Mini preparation use T7 primer for sequencing
			* subclone into ''E.coli'' Stbl4: Grow on 37°C !

ESO wikiadmin at 14:34, 30 May 2025

2025-05-30T14:34:02Z

← Older revision		Revision as of 16:34, 30 May 2025
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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
			\|}

ESO wikiadmin: Created page with "{{Protokoll-Mix|titel=Materials|Inhalt=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}}"

2025-05-30T14:33:07Z

Created page with "{{Protokoll-Mix|titel=Materials|Inhalt=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}}"

New page

{{Protokoll-Mix|titel=Materials|Inhalt=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}}

Cloning of guideRNA-Oligo into pLentiCRISPRv2 - Revision history

ESO wikiadmin at 14:35, 30 May 2025

ESO wikiadmin at 14:34, 30 May 2025

ESO wikiadmin: Created page with "{{Protokoll-Mix|titel=Materials|Inhalt=gRNA Oligos (sense/antisense; designed using ChopChop)Vector pBluescript_U6-MCS digested with BbsI (Plasmid #3468)T4 DNA ligase; 10x Ligase bufferE. coli NovaBlue}}"

ESO wikiadmin: Created page with "{{Protokoll-Mix|titel=Materials|Inhalt=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}}"