Adapt any protocols you find to suit our laboratory class.
The word count for Background and Protocol sections must be recorded after the section
Background
Lipofectamine reagents are widely used for delivery of DNA or RNA into different types of cells. Lipofectamine Reagent is a cationic lipid based transfection reagent, which is used to form liposomes that takes up the DNA. The positive charge of the liposomes and negative charge of the DNA grant the two to form a complex, then enter the cell through endocytosis. Lipofectamine reagents give high transfection efficiency and they have been used widely for many mammalian cell lines.
Materials and reagents
Lipofectamine 2000
Plasmid DNA
Pipets/tips
1.5 mL micro centrifuge tubes
Timer
24well plates (with70%-90% confluent cells)
Opti-MEM Medium
Vortex
Protocol
Before starting the experiment, spray 70% ethanol and wipe your work station. Cells should be seeded the day prior transfection so that confluency is 70-90% confluent.
1) Prepare and label 2 tubes then add 50 ul of Opti-MEM Medium to each tube.
2) Add 2ul of Lipofectamine to tube 1, and 5ul of of Lipofectamine to tube 2.
3) Mix the tubes well by vortexing.
4) Prepare a separate tube and add 250ul of Opti-MEM Medium then add 1micro gram of plasmid DNA.
5) mix it well by flicking the tube.
6) Add 50ul of diluted plasmid DNA to each tube to each and incubate them at room temperature for 5 minutes.
7) After 5 minutes add 50ul od the DNA reagent complex 1to 2 to the 24 well plate that was prepared prior.
8) Incubate for 24hr at 37 oC with 5% CO2.
9) After 24hrs assess the transfection efficiency in each well by using microscope fluorescence.
References
Invitrogen by Life Technologies. 2013. Lipofectamine® 2000 DNA Transfection Reagent Protocol. [online] Available at: https://tools.thermofisher.com/content/sfs/manuals/Lipofectamine_2000_Reag_protocol.pdf [Accessed 10 October 2020].
Cardarelli, F., Digiacomo, L., Marchini, C. et al. The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery. Sci Rep 6, 25879 (2016). [online] Available at: https://doi.org/10.1038/srep25879
[Accessed 10 October 2020].
Calcium Phosphate Transfection (To be uploaded onto moodle)
Adapt any protocols you find to suit our lab class.
The word count for Background and Protocol sections must be recorded after the section
The calcium phosphate transfection method is used to introduce DNA into several types of cells. The transfection is based on forming a calcium phosphate-DNA precipitate. Calcium phosphate induces the binding of the DNA to the cell surface. Thus, DNA then enters the cell by endocytosis. In this procedure, DNA will be mixed with a concentrated solution of CaCl2, then gets added in drops to a phosphate buffer, which forms a fine precipitate. Aeration of the phosphate buffer during the process of adding DNA-CaCl2 solution would assist with guaranteeing that the precipitate that forms is as good as could be, which is significant because clumped DNA won’t cling to or enter the cell as efficiently.
Materials and reagents
CHO-K1 cells Eppendorf tubes
Plasmid DNA 15cc tube
CaCl2 Pipets/ tips
Selection Medium timer
2X HEBS (HEPES Buffered Saline) 24well plates (with70%-90% confluent cells)
ddH2O
Protocol
Before starting, cells plating has to be done the day before, culture medium has to be replaced at least 2-5hrs prior to transfection. Cells should be seeded the day prior transfection so that confluency is 70-90% confluent.
1) In a sterile eppendorf tube add 2x HBS 200ul, ddH2O 180ul, Plasmid DNA 1ug, and 2M CaCl2 16 ul. Mix by flicking the tube.
2) Incubate at room temperature for 10 minutes.
3) After incubation mix well then add dropwise calcium phosphate/ DNA mixture to wells.
4) Incubate for 24hrs at 37 oC with 5% CO2.
5) After 24hrs check efficiency in each well by using GFP fluorescence or microscope.
Reference
Rama, K., 2016. Calcium Phosphate Transient Transfection Protocol & Guide. Scientific Methods Search Engine for Biology & Biochemistry, [online] Available at: https://scigine.com/blog/calcium-phosphate-transient-transfection/ [Accessed 10 October 2020].