Transfection of fibroblasts - Revision history

ESO wikiadmin at 16:52, 15 February 2025

2025-02-15T16:52:02Z

← Older revision		Revision as of 18:52, 15 February 2025
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	=== Materials ~~===~~		{\| class="wikitable"
	Plasmid DNA		\|+
			!Materials
	OptiMEM		\|-
			\|Plasmid DNA
	Lipofectamin 3000 or jetPrime		\|-
			\|OptiMEM
			\|-
			\|Lipofectamin 3000 or jetPrime
			\|}

	=== Procedure ===		=== Procedure ===

ESO wikiadmin at 16:44, 15 February 2025

2025-02-15T16:44:30Z

← Older revision		Revision as of 18:44, 15 February 2025
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	=== Materials ===		=== Materials ===
	Plasmid DNA		Plasmid DNA

	OptiMEM		OptiMEM

	Lipofectamin 3000 or jetPrime		Lipofectamin 3000 or jetPrime

ESO wikiadmin at 16:43, 15 February 2025

2025-02-15T16:43:54Z

← Older revision		Revision as of 18:43, 15 February 2025
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	=== Materials ===		=== Materials ===
	Plasmid DNA		Plasmid DNA

	OptiMEM		OptiMEM

	Lipofectamin 3000 or jetPrime		Lipofectamin 3000 or jetPrime

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	* ''Optional'': Medium exchange after 4h		* ''Optional'': Medium exchange after 4h

	Incubate cells at 37° C and 5% CO2		Incubate cells at 37° C and 5% CO2.

	Analyze after 24-72h.		Analyze after 24-72h.

	Lipofectamin:DNA ratio might be varied to increase transfection efficiency.		Lipofectamin:DNA ratio might be varied to increase transfection efficiency.

ESO wikiadmin at 16:43, 15 February 2025

2025-02-15T16:43:06Z

← Older revision		Revision as of 18:43, 15 February 2025
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	=== Procedure ===		=== Procedure ===
	The day before transfection: seed fibroblasts on gelatine coated 24 or 6-well plate so that they are approx. 70% confluent at time of transfection (for NIH3T3 cells 2.5x10^4 for 24 well, 1.5x10^5 for 6 well plate)		The day before transfection: seed fibroblasts on gelatine coated 24 or 6-well plate so that they are approx. 70% confluent at time of transfection (for NIH3T3 cells 2.5x10<sup>4</sup> for 24 well, 1.5x10<sup>5</sup> for 6 well plate)

	Change medium to remove and dead cells and debris		Change medium to remove and dead cells and debris

ESO wikiadmin: Created page with "=== Materials === Plasmid DNA OptiMEM Lipofectamin 3000 or jetPrime === Procedure === The day before transfection: seed fibroblasts on gelatine coated 24 or 6-well plate so that they are approx. 70% confluent at time of transfection (for NIH3T3 cells 2.5x10^4 for 24 well, 1.5x10^5 for 6 well plate) Change medium to remove and dead cells and debris Which transfection reagent works best for your cells has to be determined empirically: ==== '''JetPrime Protocol''' for..."

2025-02-15T16:42:17Z

Created page with "=== Materials === Plasmid DNA OptiMEM Lipofectamin 3000 or jetPrime === Procedure === The day before transfection: seed fibroblasts on gelatine coated 24 or 6-well plate so that they are approx. 70% confluent at time of transfection (for NIH3T3 cells 2.5x10^4 for 24 well, 1.5x10^5 for 6 well plate) Change medium to remove and dead cells and debris Which transfection reagent works best for your cells has to be determined empirically: ==== '''JetPrime Protocol''' for..."

New page

=== Materials ===
Plasmid DNA

OptiMEM

Lipofectamin 3000 or jetPrime

=== Procedure ===
The day before transfection: seed fibroblasts on gelatine coated 24 or 6-well plate so that they are approx. 70% confluent at time of transfection (for NIH3T3 cells 2.5x10^4 for 24 well, 1.5x10^5 for 6 well plate)

Change medium to remove and dead cells and debris

Which transfection reagent works best for your cells has to be determined empirically:

==== '''JetPrime Protocol''' for '''6 well''' plate (volumes for 24 well in brackets) ====

* Dilute '''2.5 µg''' (0.5 µg) DNA in '''200 µl''' (50 µl) jetprime Buffer W
* Vortex for 10 sec
* Spin down
* Add '''5 µl''' (1 µl) of jetPrime reagent
* Vortex for 10 sec
* Spin down
* Incubate 10 min at room temperature
* Add transfection mix dropwise to the cells
* Optional: Medium exchange after 6-8h

==== '''Lipofectamin protocol''' for '''6 well''' (volumes for 24 well in brackets) ====

* Dilute '''5 µl''' (1 µl) Lipofectamin 3000 reagent in '''125 µl''' (25 µl) OptiMEM
* Vortex and spin down
* Dilute '''2.5 µg''' (1µg) DNA + '''5 µl''' (2 µl) P3000 Reagent in 125 µl (25 µl) OptiMEM
* Vortex and spin down
* Add diluted DNA to diluted Lipofectamin
* Vortex and spin down
* Incubate 10-15 min at room temperature
* Add 250 µl transfection mix dropwise to the cells
* ''Optional'': Medium exchange after 4h

Incubate cells at 37° C and 5% CO2

Analyze after 24-72h.

Lipofectamin:DNA ratio might be varied to increase transfection efficiency.