Cloning of guideRNA-Oligo into pBluescript U6-MCS - Revision history

ESO wikiadmin at 14:32, 30 May 2025

2025-05-30T14:32:30Z

← Older revision		Revision as of 16:32, 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}}

	== Procedure ==		== Procedure ==
	'''gRNA Oligo annealing'''		'''gRNA Oligo annealing'''

ESO wikiadmin at 14:30, 30 May 2025

2025-05-30T14:30:42Z

← Older revision		Revision as of 16:30, 30 May 2025
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	* Ligation of vector and annealed oligos: 1 µl digested pBluescript_U6- MCS/BbsI (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)		* Ligation of vector and annealed oligos: 1 µl digested pBluescript_U6- MCS/BbsI (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), the next day 10 clones are tested via colony PCR		* Transformation in ''E.coli'' NovaBlue (plating on LB- Amp), the next day 10 clones are tested via colony PCR
	* Analysis via colony PCR forward primer No. 1371, reverse primer (antisense oligo of used gRNA pair),		*Analysis via colony PCR forward primer No. 1371, reverse primer (antisense oligo of used gRNA pair),

	<chem>-></chem>positive control: plasmid No. 3761 (pBS_U6-gRNA-mParvinB), primers #1371 + #2441; PCR product of about 230 bps; negative control: plasmid No. 3468, primers #1371 and antisense oligo of used gRNA pair; no PCR product; analysis via 2% agarose gel		<chem>-></chem> positive control: plasmid No. 3761 (pBS_U6-gRNA-mParvinB), primers #1371 + #2441; PCR product of about 230 bps; negative control: plasmid No. 3468, primers #1371 and antisense oligo of used gRNA pair; no PCR product; analysis via 2% agarose gel

	* Sequencing of positive clone: Mini preparation use T7 primer for sequencing		* Sequencing of positive clone: Mini preparation use T7 primer for sequencing

ESO wikiadmin: Created page with "== 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: {| class="wikitable" |95°C |4 minutes |- | colspan="2" |Start loop, 54x, 94°C, 1 min (-1°C/loop) |- |8°C |Store forever |} == Preparation of BbsI digested vector pBluescript_U6..."

2025-05-30T14:30:32Z

Created page with "== 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: {| class="wikitable" |95°C |4 minutes |- | colspan="2" |Start loop, 54x, 94°C, 1 min (-1°C/loop) |- |8°C |Store forever |} == Preparation of BbsI digested vector pBluescript_U6..."

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== 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:

{| class="wikitable"
|95°C
|4 minutes
|-
| colspan="2" |Start loop, 54x, 94°C, 1 min (-1°C/loop)
|-
|8°C
|Store forever
|}

== Preparation of BbsI digested vector pBluescript_U6-MCS ==

* Digestion of about 4 µg pBluescript_U6-MCS with 2 µl BbsI (10 Units in NEB buffer 2.1) in a total volume of 50 µl over night at 37° C
* Purification of vector via agarose-gel electrophoresis (expected size about 3370 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 pBluescript_U6- MCS/BbsI (about 100 ng), 1 µl annealed gRNA oligo pair, 1 µl T4 ligase, 1µl T4 ligase buffer and 6 µl ddH2O; 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), the next day 10 clones are tested via colony PCR
* Analysis via colony PCR forward primer No. 1371, reverse primer (antisense oligo of used gRNA pair),

<chem>-></chem>positive control: plasmid No. 3761 (pBS_U6-gRNA-mParvinB), primers #1371 + #2441; PCR product of about 230 bps; negative control: plasmid No. 3468, primers #1371 and antisense oligo of used gRNA pair; no PCR product; analysis via 2% agarose gel

* Sequencing of positive clone: Mini preparation use T7 primer for sequencing