Cloning of shRNA into lentiviral vector pLKO.1 - Revision history

ESO wikiadmin at 11:19, 30 May 2025

2025-05-30T11:19:35Z

← Older revision		Revision as of 13:19, 30 May 2025
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	\|Hot start		\|Hot start
	\|'''94°C'''		\|'''94°C'''
	\|~~Pause – addition of enzyme mix~~		\|4 minutes
	\|-		\|-
	\| colspan="4" \|''<small>~~7 repetitions of steps 2-4 with hybridization temperature - 1 ºC per cycle (touch-down)~~</small>''		\| colspan="4" \|''<small>Start loop, 24 cycles</small>''
	\|-		\|-
	\|2		\|2
	\|~~Denauturation~~		\|
	\|'''94°C'''		\|'''94°C'''
	\|~~20 seconds~~		\|1 minutes (-1°C/min)
	\|-		\|-
	\|3		\|3
	\|~~Hybridization~~		\| colspan="3" \|''<small>Close loop</small>''
	\|'''~~64-58°C~~'''
	~~\|20 seconds~~
	\|-		\|-
	\|4		\|4
	\|~~Elongation~~		\|
	\|'''~~72°C~~'''		\|'''70°C'''
	\|~~depending on fragment size (1 min/750 bp)~~		\|10 minutes
	\|-		\|-
	\| colspan="4" \|''<small>~~30 repetitions of steps 2-4 with hybridization temperature of 58 ºC~~</small>''		\| colspan="4" \|''<small>Start loop, ?? cycles</small>''
	\|-		\|-
	\|5		\|5
	\|~~Elongation~~		\|
	\|'''~~72°C~~'''		\|'''70°C'''
	\|5-~~10 minutes~~		\|1 min (-0.1°C/min down to 4°C)
	\|-		\|-
	\|6		\|6
	\|~~Stop~~		\| colspan="3" \|<small>Close loop</small>
	\|~~'''8°C'''~~		\|-
			\|7
			\|Store forever
			\|4°C
	\|		\|
	\|}		\|}

			=== Preparation of the lentiviral vector pLKO.1 ===

			* Double digestion of about 5 µg pLKO.1 with AgeI and EcoRI (in NEB buffer 1) over night at 37° C
			* Dephosphorylation of vector: 3-5 µg DNA, 1 µl phosphatase, 1/10 vol 10x buffer; incubation for 30 min at 37° C; heat inactivation for 10 min at 65°C
			* Purification of vector via agarose-gelelectrophoresis (expected size about 7000 bps; original size about 8900 bps)

			=== Ligation, Transformation and Analysis of Clones ===

			* Ligation reaction of vector and insert contains: 1 µl digested pLKO.1 (about 100 ng), 1 µl annealed primer, 1 µl T4 ligase, 1µl T4 ligase buffer and 6 µl ddH<sub>2</sub>O; incubation for 4 h at 4° C
			* Transformation in ''E.coli'' nova blue (plating on LB- Amp), the next day clones are picked and expanded on a new plate
			* Mini preparation (Birnboim-dooley) and subsequent analysis via agarose- gelelectrophoresis (expected size about 7000 bps, positive control plasmid No. 2512)
			* Analysis of potential positive clones via PCR: 1 µl of 1:10 diluted mini preparation, 1 µl forward primer (No. 1371), 1 µl reverse primer (No. 1372), 1 µl dNTPs, 5 µl 10x Taq-buffer, 1 µl Taq-polymerase and 40 µl ddH<sub>2</sub>O;
			* '''Positive control:''' plasmid No. 2512, PCR product of about 250 bps; negative control: plasmid No. 2521, PCR product of about 190 bps; analysis via 2% agarosegel
			* Sequencing of positive clone: Mini preparation (Qiagen; elution with EB); 1:10 dilution of primer No.1438 in EB

ESO wikiadmin at 11:14, 30 May 2025

2025-05-30T11:14:02Z

← Older revision		Revision as of 13:14, 30 May 2025
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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'''
			\|
			\|}

ESO wikiadmin: 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 ddH₂O * Annealing reaction occurs in thermocycler with the corresponding program:"

2025-05-30T11:13:37Z

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:"

New page

== 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: