jetprime DNA & sirna transfection reagent

Transcription

1 jetprime in vitro DNA & sirna Transfection Protocol Company Information... 2 Product Information Transient DNA transfection protocol Cell seeding DNA Transfection Protocol... 4 a. Overview and guidelines for any cell culture vessel format... 4 b. Detailed protocol in 6-well plates/3.5 cm dishes... 5 c. Optimization guidelines sirna transfection Cell seeding sirna transfection protocol DNA & sirna cotransfection Cell seeding DNA & sirna cotransfection protocol Stable DNA transfection Contact our Technical Assistance and Scientific Advice Service Troubleshooting Watch the video DNA Transfection using jetprime on YouTube!

2 Company Information Technical Assistance and Scientific Advice Contact the friendly Polyplus technical support via the Polyplus website: Please use the contact form. Phone: + 33 (0) Trademarks jetprime and Polyplus-transfection are registered trademarks of Polyplus-transfection. POLYPLUS-TRANSFECTION SA Bioparc Boulevard Sébastien Brant BP ILLKIRCH cedex FRANCE Phone: Fax: POLYPLUS-TRANSFECTION Inc Ave of the Americas, 34 th fl. NEW YORK, NY USA 2/12 CPT 114 vf May 2011

3 Product Information jetprime is a novel powerful molecule based on a non-liposomal formulation that ensures effective and reproducible DNA and sirna transfection into mammalian cells. jetprime is extremely efficient on a wide variety of cell lines. It is very powerful and only requires low amounts of nucleic acid per transfection; hence resulting in very low cytotoxicity. jetprime is manufactured at Polyplus-transfection. Ordering information Cat # jetprime Reagent jetprime Buffer ml 5 ml ml 60 ml ml 2 x 60 ml x 1.5 ml 10 x 60 ml C 5 x 1.5 ml 120 ml 5X Additional Buffer jetprime reagent is provided with an optimized sterile buffer (jetprime buffer). This buffer must be used to ensure successful transfection experiments. The 5X concentrated buffer provided with catalogue number C needs to be diluted 1:5 in sterile ddh2o just before use. Content 1.5 ml of jetprime transfection reagent is sufficient to perform up to 1500 transfections in 24-well plates or 375 transfections in 6-well plates. Reagent use and Limitations For research use only. Not for use in humans. Quality control Every batch of jetprime reagent is tested by DNA transfection of HeLa cells with a GFP expressing plasmid. The transfection efficiency value for each batch is indicated on the Certificate of Analysis. Formulation and Storage jetprime and its buffer are shipped at room temperature but should be stored at 4 C upon arrival to ensure long term stability. jetprime, as guaranteed and indicated on the Certificate of Analysis, will be stable for 6 months to one year when stored appropriately. 3/12 CPT 114 vf May 2011

4 1. Transient DNA 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, cells are seeded per well in 2 ml of cell growth medium 24 hours prior to transfection. For other culture formats, refer to Table 1 for the recommended number of cells to seed the day before transfection and the recommended volume to seed the cells in. jetprime is stable in the 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 seed Surface area per well (cm 2 ) medium per well to seed the cells (ml) 96-well well well well / 3.5 cm cm / flask 25 cm cm / flask 75 cm 2 1 x x cm / flask 175 cm 2 2 x x DNA Transfection Protocol a. Overview and guidelines for any cell culture vessel format 1. Dilute DNA in jetprime buffer*, vortex, spin down. 2. Add jetprime reagent, vortex, spin down. 3. Incubate for 10 min at RT. 4. Add transfection mix to the cells in cell growth medium, according to Table Change medium after 4h and analyze after 24 h. *jetprime buffer must be used for successful transfection. 4/12 CPT 114 vf May 2011

5 Table 2. DNA transfection guidelines according to the cell culture vessel per well Culture Vessel jetprime Buffer Amount of DNA (µg) jetprime reagent growth medium (ml) 96-well* well well well / 3.5 cm cm / flask 25 cm cm / flask 75 cm cm / flask 175 cm * Prepare a master mix of min 50 µl to allows accurate pipetting and homogenous preparation of the complexes Standard conditions: 1:2 DNA to jetprime ratio (w/v) i.e. for 1 µg of DNA use 2 µl of jetprime. b. Detailed protocol in 6-well plates/3.5 cm dishes 1. Dilute 2 µg DNA into 200 µl jetprime buffer (supplied). Mix by vortexing. 2. Add 4 µl jetprime, vortex for 10 s, spin down briefly. 3. Incubate for 10 min at RT. 4. Add 200 µl transfection mix per well drop wise onto the cells in regular cell growth medium, and distribute evenly. 5. Gently rock the plates back and forth and from side to side. 6. Replace transfection medium after 4 h by 4 ml of growth medium and return the plates to the incubator. 7. Analyze after 24 h or later. 5/12 CPT 114 vf May 2011

6 DNA Transfection in 6-well plates Watch the video DNA Transfection using jetprime on YouTube! 6/12 CPT 114 vf May 2011

7 c. Optimization guidelines Transfection conditions should be optimized for each tested cell line according to the conditions detailed below (Table 3). You may adjust the volume of reagent and/or the amount of DNA added per well. The amount of jetprime may range between 2 to 4 µl per µg of DNA depending on the transfected cell line (1:2, 1:3 and 1:4 DNA to jetprime ratio (w/v)). Due to the high performance of jetprime reagent, you may decrease the amount of plasmid DNA added per well. Table 3. Transfection optimization guidelines. Culture vessel jetprime Buffer Added per well Amount of DNA (µg) jetprime reagent 96-well well well well / 3.5 cm cm / flask 25 cm cm/ flask 75 cm cm/ flask 175 cm /12 CPT 114 vf May 2011

8 2. sirna transfection 2.1 Cell seeding For optimal sirna transfection conditions, we recommend using cells which are 50% confluent at the time of transfection. Typically, for experiments in 6-well plates, to cells are seeded per well in 2 ml of growth medium 24 hours prior to transfection. For other culture formats, refer to Table 4 for the recommended number of cells to seed the day before transfection and the recommended volume to seed the cells in. jetprime is stable in the presence of serum and antibiotics therefore you may use serum and antibiotic containing medium during the entire experiment. Table 4. Recommended number of cells to seed the day before transfection. Culture vessel Number of adherent cells to seed Surface area per well (cm 2 ) medium per well to seed the cells (ml) 24-well well well / 3.5 cm cm / flask 25 cm cm / flask 75 cm x x cm / flask 175 cm 2 2 x x /12 CPT 114 vf May 2011

9 2.2 sirna transfection protocol For optimal sirna-mediated silencing, we recommend using nm sirna. The following conditions are given per well in a 6 well plate. For other culture format, please refer to Table Dilute 22 to 110 pmoles sirna (for a final concentration of 10 to 50 nm per well) into 200 µl of jetprime buffer. Mix by pipeting up and down. 2. Add 4 µl jetprime reagent, vortex for 10 sec, spin down briefly. 3. Incubate for 10 to 15 min at RT. 4. Add the transfection mix to the cells in regular cell growth medium drop wise. 5. Gently rock the plate back and forth and return the plate to the incubator. 6. Replace transfection medium by cell growth medium 24 hours after transfection and analyse as required. Table 5. sirna Transfection guidelines according to the cell culture vessel. Culture vessel Amount of sirna (pmoles) 10 nm Amount of sirna (pmoles) 50 nm jetprime reagent jetprime Buffer for complex formation growth medium per well Final volume in the well 24-well µl 550 µl 12-well ml 1.1 ml 6-well / 3.5 cm ml 2.2 ml 6 cm/ flask 25 cm ml 4.2 ml 10 cm/ flask 75 cm ml 10.5 ml 9/12 CPT 114 vf May 2011

10 3. DNA & sirna cotransfection 3.1 Cell seeding For optimal transfection conditions, we recommend using cells which are 60 to 80% confluent at the time of transfection. Typically, for experiments in 6-well plates, to cells are seeded per well 24 hours prior to transfection. For other culture formats, refer to Table 1 for the recommended number of cells to seed the day before transfection and the recommended volume to seed the cells in. jetprime is stable in the presence of serum and antibiotics therefore you may use serum and antibiotic containing medium during the entire experiment. 3.2 DNA & sirna cotransfection protocol For DNA/siRNA cotransfection experiment, we recommend using 400 ng DNA and 10 to 50 nm sirna per well in a 6-well plate. The following conditions are given per well of a 6 well plate. For other culture format, please refer to Table Dilute 400 ng of DNA and 22 to 110 pmoles sirna (final concentration: 10 to 50 nm) into 200 µl of jetprime buffer. Mix by pipeting up and down. 2. Add 4 µl jetprime reagent, vortex for 10 sec, spin down briefly. 3. Incubate for 10 to 15 min at RT. 4. Add the transfection mix to the cells still in cell growth medium drop wise. 5. Gently rock the plate back and forth and return the plate to the incubator. 6. Replace transfection medium by cell growth medium 24 hours after transfection and analyze as required. 10/12 CPT 114 vf May 2011

11 Table 6. DNA & sirna cotransfection guidelines according to the cell culture vessel. Culture vessel Amount of DNA Amount of sirna (pmoles) 10 nm Amount of sirna (pmoles) 50 nm jetprime reagent jetprime Buffer for complex formation growth medium on the cells Final volume in the well 24-well ng µl 550 µl 12-well ng ml 1.1 ml 6-well / 3.5 cm 400 ng ml 2.2 ml 6 cm/ 2 flask 25 cm 800 ng ml 4.2 ml 10 cm/ 2 flask 75 cm 2 µg ml 10.5 ml 4. Stable DNA transfection jetprime TM is suitable for stable DNA transfection. Perform stable transfection in 6-well plates, 60 mm or 10 cm dishes. 1. If needed, linearize plasmid DNA construct encoding for antibiotic selection. 2. Perform transfection as described in the standard protocol in Section Start antibiotic selection h after transfection. 4. Maintain antibiotic selection as long as required. 5. Check for integration of the plasmid DNA or stable expression of your protein of expression. Contact our Technical Assistance and Scientific Advice Service Contact the friendly Polyplus technical support via: The Polyplus website: support@polyplus-transfection.com Phone: + 33 (0) /12 CPT 114 vf May 2011

12 Troubleshooting Observations Actions Optimize the volume of jetprime reagent and the amount of plasmid DNA added per well. Increase the volume of jetprime reagent first; if insufficient, increase the amount of DNA according to Table 3. Use high-quality plasmid preparation, free of proteins and RNA (OD 260/280 > 1.8). Low DNA transfection efficiency Low sirna mediated silencing efficiency Cellular toxicity Use a plasmid containing a common reporter gene such as Luciferase or GFP as positive control. Preferably use a DNA preparation at a concentration of 0.3 to 1 µg/µl. For cells known to be difficult to transfect, start by using double the amount of DNA than suggested in Table 2. Then decrease DNA amount. Ensure that jetprime reagent was diluted in jetprime buffer included and not in any other buffer. Ensure that transfection was performed in presence of serum since jetprime gives higher transfection efficiency in presence of serum. Ensure transfection is not performed in OptiMEM Ensure that all reagents are RNAse free. Ensure of the quality of your sirna (desalted, concentration, annealing, design). Optimize the amount of sirna used and of DNA if performing cotransfection. Analyze transfection at an earlier time point e.g. at 24 h instead of 48 h. Wash cells 4 h after transfection Decrease the amount of plasmid DNA added per well Ensure that jetprime reagent was diluted in jetprime buffer included. Decrease the amount of jetprime reagent. Ensure that the plasmid preparation is endotoxin-free. Verify the toxicity of the expressed protein. If the expressed protein is toxic for the cells, reduce the amount of plasmid DNA. Ensure that transfection was performed in presence of serum since jetprime gives higher transfection efficiency in presence of serum. Ensure transfection is not performed in OptiMEM 12/12 CPT 114 vf May 2011