jetPRIME转染试剂说明_精品文档.pdf

1、 Polyplus-transfection S.A.-Bioparc-850 Bd S.Brant-67400 Illkirch-France-Phone:+33 3 90 40 61 80-Fax:+33 3 90 40 61 81 Polyplus-transfection Inc.-1251 Ave of the Americas-34th fl.-New-York-NY 10020-USA www.polyplus- jetPRIME in vitro DNA&siRNA transfection reagent PROTOCOL DESCRIPTION jetPRIME is a

2、novel powerful molecule based on a polymer formulation manufactured at Polyplus-transfection.jetPRIME ensures effective and reproducible DNA and siRNA transfection into mammalian cells.jetPRIME is extremely efficient on a wide variety of cell lines.This powerful reagent only requires low amounts of

3、nucleic acid per transfection,hence resulting in very low cytotoxicity.1 Transient DNA transfection protocol.2 1.1 Cell seeding.2 1.2 DNA transfection protocol.2 1.3 Virus production in adherent cells.5 1.4 Optimization guidelines.5 2 siRNA transfection protocol.6 2.1 Cell seeding.6 2.2 siRNA transf

4、ection protocol.7 3 DNA&siRNA cotransfection protocol.7 3.1 Cell seeding.7 3.2 DNA&siRNA cotransfection protocol.8 4 Transfection of CRISPR/Cas9 .9 5 Stable DNA transfection.9 6 Troubleshooting.10 7 Product information.11 CPT114vJ(March 2015)2 jetPRIME DNA&siRNA Transfection Reagent 1 TRANSIENT DNA

5、TRANSFECTION PROTOCOL 1.1 CELL SEEDING For optimal DNA transfection conditions,we recommend using cells which are 60 to 80%confluent at the time of transfection.Typically,for experiments in 6-well plates,200 000 cells are seeded per well in 2 ml of cell growth medium 24 h prior to transfection.For o

6、ther culture formats,refer to Table 1.jetPRIME is stable in presence of serum and antibiotics therefore you may use serum and antibiotic containing medium during the entire experiment.Table 1.Recommended number of cells to seed the day before transfection.Culture vessel Number of adherent cells to s

7、eed Surface area per well(cm2)Volume of medium per well to seed the cells(ml)96-well 7 500-10 000 0.3 0.1 24-well 50 000-80 000 1.9 0.5 12-well 80 000-150 000 3.8 1 6-well/35 mm 150 000-250 000 9.4 2 60 mm/flask 25 cm2 250 000-800 000 25-28 5 100 mm/flask 75 cm2 1 x 106-2 x 106 75-78.5 10 150 mm/fla

8、sk 175 cm2 2 x 106-5 x 106 153-175 20 1.2 DNA TRANSFECTION PROTOCOL The following conditions are given per well of a 6-well plate.For other culture format,please refer to Table 2.1.Dilute 2 g DNA into 200 l jetPRIME buffer(supplied).Mix by vortexing.2.Add 4 l jetPRIME,vortex for 10 s,spin down brief

9、ly.3.Incubate for 10 min at RT.4.Add 200 l of transfection mix per well drop wise onto the cells in serum containing medium,and distribute evenly.5.Gently rock the plates back and forth and from side to side.6.If needed,replace transfection medium after 4 h by cell growth medium and return the plate

10、s to the incubator.7.Analyze after 24 h or later.Polyplus-transfection S.A.-Bioparc-850 Bd S.Brant 67400 Illkirch France-Phone:+33 3 90 40 61 80-Fax:+33 3 90 40 61 81 Polyplus-transfection Inc.-1251 Ave of the Americas-34th fl.-New-York-NY 10020-USA www.polyplus- 3 jetPRIME DNA&siRNA Transfection Re

11、agent Table 2.DNA transfection guidelines according to the cell culture vessel per well Culture Vessel Volume of jetPRIME Buffer(l)Amount of DNA(g)Volume of jetPRIME reagent(l)Volume of growth medium(ml)96-well*5 0.1 0.2-0.3 0.1 24-well 50 0.5 1-1.5 0.5 12-well 75 0.8 1.6-2.4 1 6-well/35 mm 200 2 4-

12、6 2 60 mm/flask 25 cm2 200 4 8-12 5 100 mm/flask 75 cm2 500 10 20-30 10 150 mm/flask 175 cm2 1000 20 40-60 20*Prepare a master mix of minimum 50 l to allow accurate pipetting and homogenous preparation of the complexes NOTE:jetPRIME buffer must be used for successful transfection.Browse our cell tra

13、nsfection database to find the optimized conditions according to your cell line.http:/www.polyplus- the video“DNA Transfection using jetPRIME”on YouTube!http:/ Standard conditions 1:2 DNA to jetPRIME ratio(w/v)for 1 g of DNA use 2 l of jetPRIME CPT114vJ(March 2015)4 jetPRIME DNA&siRNA Transfection R

14、eagent DNA Transfection in 6-well plates Polyplus-transfection S.A.-Bioparc-850 Bd S.Brant 67400 Illkirch France-Phone:+33 3 90 40 61 80-Fax:+33 3 90 40 61 81 Polyplus-transfection Inc.-1251 Ave of the Americas-34th fl.-New-York-NY 10020-USA www.polyplus- 5 jetPRIME DNA&siRNA Transfection Reagent 1.

15、3 VIRUS PRODUCTION IN ADHERENT CELLS jetPRIME is ideal for virus production,especially retrovirus,AAV and lentivirus,in adherent cells(ex:HEK-293T).For cotransfection of multiple plasmids,the total DNA amount per well/plate should not exceed the DNA amount indicated in Table 2.The ratio to use for e

16、ach plasmid depends on the size of the plasmids,the plasmid constructs and the desired expression level of each plasmid.Please adjust the ratios according to your application.Each plasmid should represent at least 10%of the total DNA amount per well/plate.The following conditions are given per 100 mm dish.For other culture format,please refer to Table 2.For optimization,please refer to Table 3.1.Dilute 10 g total DNA amount into 500 l jetPRIME buffer(supplied).Mix by vortexing.2.Add 20 l jetPRIM