QuickShuttle Transfection Reagent

Transcription

1 QuickShuttle Transfection Reagent General Description QuickShuttle is a proprietary cationic polymer-based transfection reagent, which is optimized for the purpose of maximal transfection efficiency, ease of use, and minimal cytotoxicity. It is recommended for plasmid DNA transfection into mammalian cells by means of transient transfection as well as stable cell line generation. QuickShuttle has two unique features that other conventional transfection reagents don t have: (1) transfection could be done immediately after cell subculture; (2) transfection could be completed in just one minute. User Guidelines 1. With QuickShuttle transfection could be done immediately after cell subculture so that the 18~24 hours of waiting time before transfection could be saved. 2. With QuickShuttle transfection could be completed in just one minute. The 10~30 minutes of incubation time could be saved when prepare DNA/transfection reagent complexes. And transfection could be performed in the presence of bovine serum and antibiotics without the compromise of transfection efficiency, which makes no need to change media before and after transfection. 3. With the combination of features of transfection immediately after cell subculture, one-minute transfection and high transfection efficiency, some transfection experiments could be finished with 24 hours after cell passage, which could save hours compared with other conventional transfection reagents. 4. For transfection in 24-well plates, we recommend the amounts of endotoxin-free plasmid DNA and QuickShuttle are in the ranges of 1~2µg and 3~5µl per well, respectively, which should be optimized with reporter genes according to specific cells and media used if best results are expected. 5. QuickShuttle has been tested with DMEM RPMI-1640 and M199 media. Although we recommend using DMEM in our protocols, available data showed that M199 could enhance the transfection of Vero and 293FT cells compared with the other two media. Therefore, transfection parameters could be optimized using different media when necessary. 1

2 6. QuickShuttle has been designed for the transfection of conventional mammalian cell lines. We don t recommend but user could try it on primary as well as diploid mammalian cells and cells from other species such as insect cells. 7. QuickShuttle is recommended to be shipped at ambient temperature and stored at 2~8ºC (stable up to 18 months at this temperature). However, it is highly stable at any temperature ranging from -30ºC to room temperature. Product Name: QuickShuttle -Basic (Basic QuickShuttle Transfection Reagent) Cat No: KX Size: 0.8 ml Intended use: transient and stable transfection of most mammalian adherent cell lines. Transfection Guidelines: 1. Plasmid DNA: prepared with low endotoxin or endotoxin-free plasmid extraction kit. 2. Diluent: 0.85%(W/V)saline,prepared with low endotoxin pure water,sterilized by autoclave or 0.22µm filtration. 3. Media: tested with DMEM RPMI-1640 and M199, recommend to use DMEM with 5-10% bovine serum, and transfection efficiency could be optimized using other media. 4. For transfection in 24-well plates, we recommend the amounts of endotoxin-free plasmid DNA and QuickShuttle are 2µg and 3~5µl per well, respectively, which should be 2

3 optimized with reporter genes according to specific cells and media used if best results are expected. 5. For stable cell line generation, transfection procedure could be simplified as cells are transfected immediately after subculture, saving as long as 18~24 hours of waiting time compared with other conventional transfection reagents. Transfection Protocol: 1. 18~24 hours prior to transfection, plate 5~10 x 10 4 cells per well into 24-well plates in 1 ml of complete medium. Note: For stable cell line generation, transfection procedure could be simplified as cells are transfected immediately after subculture, saving as long as 18~24 hours of waiting time compared with other conventional transfection reagents. 2. Dilute 2µg of endotoxin-free plasmid DNA and 3~5µl of QuickShuttle respectively into 50µl of 0.85% (w/v) sterilized saline. Note: The dosage of transfection reagent should be optimized according to specific cells and media used, which is theoretically within the range of 3~5µl per well. 3. Combine two solutions and mix well by pipetting or flicking. Note: The 10~30 minutes of incubation time in conventional transfection experiments could be saved when prepare DNA/transfection reagent complexes. 4. Add the DNA/transfection reagent complexes directly into culture media, and mix gently by pipetting or rocking the plate back and forth. Note: Transfection could be performed in the presence of bovine serum and antibiotics without the compromise of transfection efficiency. In rare cases if cell detachment occurs, please remove 500µl of medium from the culture to dilute the DNA/transfection reagent complexes then transfer back to the culture. 5. Transfer 24-well plates to a 37ºC/5%CO 2 incubator. Note: It s unnecessary to change media after 4~6 hours of incubation. 6. Perform transient expression analysis or stable cell line selection using antibiotics 24~72 hours post-transfection. Product Name: QuickShuttle -Enhanced (Enhanced QuickShuttle Transfection Reagent) 3

4 Cat No:KX Size: 0.8 ml Intended use: transient and stable transfection of difficult-to-be transfected mammalian adherent cell lines. Transfection Guidelines: 1. Plasmid DNA: prepared with low endotoxin or endotoxin-free plasmid extraction kit. 2. Diluent: 0.85%(W/V)saline,prepared with low endotoxin pure water,sterilized by autoclave or 0.22µm filtration. 3. Media: tested with DMEM RPMI-1640 and M199, recommend to use DMEM with 5-10% bovine serum, and transfection efficiency could be optimized using other media. 4. For transfection in 24-well plates, we recommend the amounts of endotoxin-free plasmid DNA and QuickShuttle are in the ranges of 1~2µg and 3~5µl per well, respectively, which should be optimized with reporter genes according to specific cells and media used if best results are expected. 5. For stable cell line generation, transfection procedure could be simplified as cells are transfected immediately after subculture, saving as long as 18~24 hours of waiting time compared with other conventional transfection reagents. Transfection Protocol: 1. 18~24 hours prior to transfection, plate 5~10 x 10 4 cells per well into 24-well plates in 1 ml of complete medium. 4

5 Note: For stable cell line generation, transfection procedure could be simplified as cells are transfected immediately after subculture, saving as long as 18~24 hours of waiting time compared with other conventional transfection reagents. 2. Dilute 1~2μg of endotoxin-free plasmid DNA and 3~5µl of QuickShuttle respectively into 50µl of 0.85% (w/v) sterilized saline. Note: The dosage of plasmid DNA and transfection reagent should be optimized according to specific cells and media used, which are theoretically within the ranges of 1~2µg and 3~5µl per well, respectively. 3. Combine two solutions and mix well by pipetting or flicking. Note: The 10~30 minutes of incubation time in conventional transfection experiments could be saved when prepare DNA/transfection reagent complexes. 4. Add the DNA/transfection reagent complexes directly into culture media, and mix gently by pipetting or rocking the plate back and forth. Note: Transfection could be performed in the presence of bovine serum and antibiotics without the compromise of transfection efficiency. In rare cases if cell detachment occurs, please remove 500µl of medium from the culture to dilute the DNA/transfection reagent complexes then transfer back to the culture. 5. Transfer 24-well plates to a 37ºC/5%CO 2 incubator. Note: It s unnecessary to change media after 4~6 hours of incubation. 6. Perform transient expression analysis or stable cell line selection using antibiotics 24~72 hours post-transfection. Product Name: QuickShuttle -Superfast (Superfast QuickShuttle Transfection Reagent) 5

6 Cat No:KX Size: 0.8 ml Intended use: (1) transient and stable transfection of most mammalian adherent cell lines (transfection immediately after cell subculture); (2) stable transfection of most mammalian suspension cell lines. Transfection Guidelines: 1. Plasmid DNA: prepared with low endotoxin or endotoxin-free plasmid extraction kit. 2. Diluent: 0.85%(W/V)saline,prepared with low endotoxin pure water,sterilized by autoclave or 0.22µm filtration. 3. Media: tested with DMEM RPMI-1640 and M199, recommend to use DMEM with 5-10% bovine serum, and transfection efficiency could be optimized using other media. 4. For transfection in 24-well plates, we recommend the amounts of endotoxin-free plasmid DNA and QuickShuttle are in the ranges of 1~2µg and 3~5µl per well, respectively, which should be optimized with reporter genes according to specific cells and media used if best results are expected. 5. Not recommended for adherent cell lines with low adherence ability, such as various 293 cell lines. 6. This reagent is able to transfect most mammalian suspension cell lines, although at low efficiency but sufficient for stable cell line generation after antibiotic selection. Transfection Protocol: 6

7 1. Plate 1~2 x 10 5 suspension or freshly digested adherent cells per well into 24-well plates in 1 ml of complete medium. Note: Transfection could be performed immediately after cell subculture, saving as long as 18~24 hours of waiting time compared with other conventional transfection reagents. 2. Dilute 1~2μg of endotoxin-free plasmid DNA and 3~5µl of QuickShuttle respectively into 50µl of 0.85% (w/v) sterilized saline. Note: The dosage of plasmid DNA and transfection reagent should be optimized according to specific cells and media used, which are theoretically within the ranges of 1~2µg and 3~5µl per well, respectively. 3. Combine two solutions and mix well by pipetting or flicking. Note: The 10~30 minutes of incubation time in conventional transfection experiments could be saved when prepare DNA/transfection reagent complexes. 4. Add the DNA/transfection reagent complexes directly into culture media, and mix gently by pipetting or rocking the plate back and forth. Note: Transfection could be performed in the presence of bovine serum and antibiotics without the compromise of transfection efficiency. In rare cases if cell detachment occurs, please remove 500µl of medium from the culture to dilute the DNA/transfection reagent complexes then transfer back to the culture. 5. Transfer 24-well plates to a 37ºC/5%CO 2 incubator. Note: It s unnecessary to change media after 4~6 hours of incubation. 6. Perform transient expression analysis or stable cell line selection using antibiotics 24~72 hours post-transfection. 7

8 Product Name: QuickShuttle -293 (QuickShuttle Transfection Reagent for 293 Cells) Cat No:KX Size: 0.8 ml Intended use: transient and stable transfection of various 293 cell lines (transfection immediately after cell subculture). Transfection Guidelines: 1. Plasmid DNA: prepared with low endotoxin or endotoxin-free plasmid extraction kit. 2. Diluent: 0.85%(W/V)saline,prepared with low endotoxin pure water,sterilized by autoclave or 0.22µm filtration. 3. Media: tested with DMEM RPMI-1640 and M199, recommend to use DMEM with 5-10% bovine serum, and transfection efficiency could be optimized using other media. 4. For transfection in 24-well plates, we recommend the amounts of endotoxin-free DNA and QuickShuttle are 2µg and 3~5µl per well, respectively, which should be optimized with reporter genes according to specific 293 cell lines and media used if best results are expected. Transfection Protocol: 1. Plate 1~2 x 10 5 freshly digested 293 cells per well into 24-well plates in 1 ml of complete medium. Note: Transfection could be performed immediately after cell subculture, saving as long as 18~24 hours of waiting time compared with other conventional transfection reagents. 8

9 2. Dilute 2μg of endotoxin-free plasmid DNA and 3~5µl of QuickShuttle respectively into 50µl of 0.85% (w/v) sterilized saline. Note: The dosage of transfection reagent should be optimized according to specific 293 cell lines and media used, which is theoretically within the range of 3~5µl per well. 3. Combine two solutions and mix well by pipetting or flicking. Note: The 10~30 minutes of incubation time in conventional transfection experiments could be saved when prepare DNA/transfection reagent complexes. 4. Add the DNA/transfection reagent complexes directly into culture media, and mix gently by pipetting or rocking the plate back and forth. Note: Transfection could be performed in the presence of bovine serum and antibiotics without the compromise of transfection efficiency. In rare cases if cell detachment occurs, please remove 500µl of medium from the culture to dilute the DNA/transfection reagent complexes then transfer back to the culture. 5. Transfer 24-well plates to a 37ºC/5%CO 2 incubator. Note: It s unnecessary to change media after 4~6 hours of incubation. 6. Perform transient expression analysis or stable cell line selection using antibiotics 24~72 hours post-transfection. Product Name: QuickShuttle -Hela (QuickShuttle Transfection Reagent for Hela Cells) Cat No:KX Size: 0.8 ml 9

10 Intended use: transient and stable transfection of Hela cells (transfection immediately after cell subculture). Transfection Guidelines: 1. Plasmid DNA: prepared with low endotoxin or endotoxin-free plasmid extraction kit. 2. Diluent: 0.85%(W/V)saline,prepared with low endotoxin pure water,sterilized by autoclave or 0.22µm filtration. 3. Media: tested with DMEM RPMI-1640 and M199, recommend to use DMEM with 5-10% bovine serum, and transfection efficiency could be optimized using other media. 4. For transfection in 24-well plates, we recommend the amounts of endotoxin-free plasmid DNA and QuickShuttle are in the ranges of 1~2µg and 3~5µl per well, respectively, which should be optimized with reporter genes according to media used if best results are expected. Transfection Protocol: 1. Plate 1~2 x 10 5 freshly digested Hela cells per well into 24-well plates in 1 ml of complete medium. Note: Transfection could be performed immediately after cell subculture, saving as long as 18~24 hours of waiting time compared with other conventional transfection reagents. 2. Dilute 1~2μg of endotoxin-free plasmid DNA and 3~5µl of QuickShuttle respectively into 50µl of 0.85% (w/v) sterilized saline. Note: The dosage of plasmid DNA and transfection reagent should be optimized according to media used, which are theoretically within the ranges of 1~2µg and 3~5µl per well, respectively. 3. Combine two solutions and mix well by pipetting or flicking. Note: The 10~30 minutes of incubation time in conventional transfection experiments could be saved when prepare DNA/transfection reagent complexes. 4. Add the DNA/transfection reagent complexes directly into culture media, and mix gently by pipetting or rocking the plate back and forth. Note: Transfection could be performed in the presence of bovine serum and antibiotics without the compromise of transfection efficiency. In rare cases if cell detachment occurs, please remove 500µl of medium from the culture to dilute the DNA/transfection reagent complexes then transfer back to the culture. 5. Transfer 24-well plates to a 37ºC/5%CO 2 incubator. 10

11 Note: It s unnecessary to change media after 4~6 hours of incubation. 6. Perform transient expression analysis or stable cell line selection using antibiotics 24~72 hours post-transfection. 11