DeliverX™ and DeliverX Plus siRNA Transfection Kits
Contents
1. Page 12 DeliverX and DeliverX Plus Kit User Manual Transfecting Cells in Suspension Transfecting Cells in Suspension About Transfecting Cells in Suspension Assay Preparation Procedure This procedure describes the transfection of adherent cells after trypsinization and while they are in suspension Transfect adherent cells in suspension when you want to do the treatment and transfection in the same day The same procedure can also be used for suspension cell types Note In some cases this procedure improves the knockdown efficiency of adherent cell types Pre warm both serum free and complete growth media to 37 C Prepare adherent cells in suspension if using by trypsinizing the cells using your normal procedure and suspend them in complete growth media When performing transfections in different sized wells scale the amount of reagents in proportion to the surface area of the plate well or dish See Scaling Transfections on page 14 for more information To transfect cells in suspension in a 6 well format Step Action 1 Pellet the cells in a 15 mL tube by spinning for 5 minutes at 400 x g 2 Gently aspirate the media avoiding cell loss and resuspend the cells in 5 mL of PBS and spin again at 400 x g for 5 minutes 3 Gently aspirate the PBS avoiding cell loss and resuspend the cells in PBS to a concentration of 0 3 0 9 x 106 cells mL 4 Dispense 1 mL aliquots into 2 mL microtube2. cells mL Required Well Complex Media Growth Media 6 well plate 0 3 0 9 x 106 1 000 uLa 300 uL 300 uL 1 000 uL 12 well plate 0 1 0 4 x 106 1 000 uLa 150 uL 150 uL 500 uL 24 well plate 5 x 106 30 uLb 75 uL 75 uL 250 uL 96 well plate 1 x 106 10 uLb 30 uL 30 uL 100 uL a Spin cells down in a microtube remove PBS and resuspend cells with the working transfection complex After adding the serum free media transfer the contents of the microtube to a single well of a 6 well or 12 well plate as appropriate Add complete growth media b Add cell suspensions directly to the cell culture plate Perform all subsequent steps in the cell culture plate DeliverX and DeliverX Plus Kit User Manual Page 15 Troubleshooting Troubleshooting Problems and Recommended To troubleshoot transfections using DeliverX and DeliverX Plus kits Actions Page 16 Observation Possible Cause Recommended Action RNA knockdown not as expected Denatured siRNA Do not use water to dilute the siRNA which can cause denaturation of siRNA Use 1X TE pH 8 0 to dilute stock siRNA to 5 uM and DeliverX siRNA Buffer 1 to dilute siRNA during complex formation Poorly designed siRNA Make sure the sequence of the siRNA is correct for the gene of interest More than one sequence may need to be tested for optimal knockdown efficiency Cell density not optimal Evaluate cell densities outside of the recommended 40 80 confluence a
3. cycles Store components at recommended temperatures Product shelf life is 6 months from date of shipment if stored properly CAUTION All chemicals should be considered potentially hazardous We recommend that this product and its components be handled by those trained in laboratory techniques and be used according to the principles of good laboratory practice For research use only Do not use internally or externally in humans or animals Required and Recommended Materials Not Provided Materials Required But Not Provided Page 6 Item Source Cell culture reagents and equipment Major laboratory suppliers MLS Tris EDTA buffer TE pH 8 0 P N 9849 Ambion Inc Phosphate buffered saline pH 7 2 PBS P N 70013 032 Invitrogen siRNA for silencing target of interest MLS mRNA and or protein detection method following siRNA knockdown Panomics Inc for mRNA quantitation P N QG0001 Ultrasonic cleaner bath sonicator with 30 40 kHz sonication Panomics Inc P N DX0400 or equivalent DeliverX and DeliverX Plus Kit User Manual Assay Workflow and Recommended Guidelines Controls and Optimization Materials Item Source Recommended But Not Provided QuantiGene Reagent System for mRNA Panomics Inc P N QG0001 quantitation FAM labeled siRNA control Panomics Inc P N DX0100 GAPDH siRNA control human Panomics Inc P N DX0101 Negative s
4. reagents in proportion to the surface area of the plate well or dish See Scaling Transfections on page 14 for more information To transfect adherent cells grown in 96 well plates Step Action 1 Carefully remove media from the wells and wash once with 100 uL well of PBS 2 Add 30 uL well of the working siRNA siRNA transfection reagent complex IMPORTANT Manually rock do not swirl the plate to evenly distribute the complex over the well surface 3 Incubate at room temperature for 3 5 minutes 4 Add 30 uL well of serum free media 5 Incubate under normal cell culture conditions typically at 37 C and 5 CO for 2 4 hours Note For maximum knockdown we recommend 4 hours 6 Add 100 uL well of complete growth media At this step the final concentration of siRNA in the sample is 30 nM Note For cells that require a precise serum level you may have to adjust the serum concentration 7 Incubate under normal cell culture conditions typically at 37 C and 596 CO for 24 hours or desired time intervals Note 24 48 hour incubation times are typical when measuring mRNA knockdown 48 72 hour incubation times are typical when measuring protein knockdown For longer incubation times you may need to change the media 24 hours post transfection 8 Measure mRNA and or protein knockdown as appropriate We recommend quantitating mRNA expression levels with the QuantiGene Reagent System
5. the application or use of this information Contents DeliverX and DeliverX Plus siRNA Transfection Kit and Manual Overview 5 About the Kite i21 oV o OR ete rise 5 About the Manual 0000 c eee rr 5 DeliverX and DeliverX Plus siRNA Transfection Kit Contents and Storage 6 Contents and Storage of DeliverX Kit 0 0 llle 6 Contents and Storage of DeliverX Plus Kit 0 0000 a eee eae 6 Storage Recommendations 0 000 eee eee 6 Safety Warnings and Precautions oooooccoococoooc eae 6 Required and Recommended Materials Not Provided 0 000 ce ewes 6 Materials Required But Not Provided 2000 ccc ee eeeeeee 6 Materials Recommended But Not Provided 200 02005 7 Assay Workflow and Recommended Guidelines Controls and Optimization 7 Assay WOIKIOW said d o EAR heh ta 7 Transfection Guidelines 00 000 cee tees 7 Recommended Controls 0000 ete 8 Optimizing Transfections ara raad a e a eee 8 Preparing the Cells the Day Before Transfection anuanua aaan 9 Adherent Cell Types roere mane anae a m a hts 9 Suspension Cells or Adherent Cells in Suspension 9 Preparing the siRNA siRNA Transfection Reagent Complex 240 10 About Preparing the Complex 0000 ernennen een eee 10 Pr ced re copa a lt u adie RE ERA RE ants eel a LS 10 Transfecti
6. DeliverX and DeliverX Plus siRNA Transfection Kits User Manual Panomics P N 10586 060504 Rev A Panomics Inc DeliverX and DeliverX Plus siRNA Transfection Kit User Manual Copyright Copyright 2006 Panomics Inc All rights reserved Trademarks DeliverX is a trademark of Panomics Inc QuantiGene is a registered trademark exclusively licensed to Panomics Inc All other trademarks belong to their respective owners Citing DeliverX in Publications When describing a procedure for publication using this product we would appreciate it if you would refer to it as the DeliverXTM siRNA Transfection Kit or DeliverX Plus siRNA Transfection Kit If a paper cites a DeliverX product and is published in a research journal the lead author s may receive a travel stipend for use at a technology conference or tradeshow by sending a copy of the paper to our technical support group at techsupport panomics com or via fax at 510 818 2610 Licenses DeliverX and DeliverX Plus transfection reagents are manufactured and distributed by Panomics under license from CNRS France Disclaimer Panomics Inc reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Panomics Inc assumes no liability for any errors or omissions nor for any damages resulting from
7. NA Transfection Reagent Complex Preparing the siRNA siRNA Transfection Reagent Complex About Preparing Sonication of the DeliverX and DeliverX Plus Transfection Reagents per the the Complex procedure below is critical for proper formation of the complex used for transfection Once the complex has been properly formed it is amenable to dilution for evaluating a range of siRNA delivery concentrations This novel peptide based delivery system requires no optimization of the siRNA to transfection reagent ratio and enables high efficiency siRNA transfection typically with 30 nM or less Procedure The following procedure prepares transfection complexes sufficient for the transfection of 13 wells of a 96 well plate 4 wells of a 24 well plate 2 wells of a 12 well plate or 1 well of a 6 well plate When scaling up the number of transfections to be performed or when performing transfections in different sized wells the amount of reagents should be scaled in proportion to the surface area of the plate well or dish see Scaling Transfections on page 14 for more information IMPORTANT We do not recommend scaling up reagent volumes greater than 3X when preparing the working siRNA siRNA transfection reagent complexes Instead we recommend that multiple replicate working transfection complexes be prepared in parallel then combined into a single tube to produce a bulk volume of working transfection complex To prepare DeliverX or DeliverX Plus siR
8. NA transfection reagent complex Step Action 1 Thaw siRNAs and DeliverX or DeliverX Plus Transfection Reagent and store on ice 2 Prepare working stocks of siRNAs by diluting the siRNAs to 5 uM with TE buffer 3 Dilute the 5 uM siRNA working stocks with Buffer 1 in 1 5 mL tubes as described in the table below This preparation will yield a final siRNA delivery concentration of 30 nM 5 uM siRNA 5 uM Negative Working Control siRNA Sample Stocks uL uL Buffer 1 uL Tube 1 Target siRNA 19 u d Tube 2 Negative 13 37 Control siRNA Tube 3 Cells only B PO a Recommended control not supplied with the kit Page 10 DeliverX and DeliverX Plus Kit User Manual Preparing the siRNA siRNA Transfection Reagent Complex To prepare DeliverX or DeliverX Plus siRNA transfection reagent complex continued Step Action 4 Prepare the DeliverX or the DeliverX Plus Transfection Reagent a Sonicate the DeliverX or DeliverX Plus siRNA Transfection Reagent at maximum output and continuous power for 3 5 minutes to achieve a homogenous solution b Prepare dilutions in 1 5 mL tubes as described in the table below c After diluting vortex briefly and sonicate again at maximum output and continuous power for 3 5 minutes IMPORTANT Sonication of DeliverX or DeliverX Plus siRNA Transfection Reagent is critical for achieving good complex formation Ensure that the tubes are submerg
9. a MPG technology uses virus derived amphipathic peptides that directly interact with nucleic acid cargos to form non covalent nanoparticles 150 200 nm capable of diffusing through the plasma membrane and releasing their contents inside the cell The mechanism of entry is receptor independent involves MPG lipid interactions and avoids the endocytic pathway thereby preventing endosomal or lysosomal degradation of cargos The manual contains a description of the kit contents and guidelines recommendations and procedures for performing transfections using the DeliverX siRNA and DeliverX Plus siRNA Kits DeliverX and DeliverX Plus Kit User Manual Page 5 DeliverX and DeliverX Plus siRNA Transfection Kit Contents and Storage DeliverX and DeliverX Plus siRNA Transfection Kit Contents and Storage Contents and Storage of DeliverX Kit Contents and Storage of DeliverX Plus Kit Storage Recommendations Safety Warnings and Precautions DeliverX siRNA Transfection Kit Contents Component Storage DeliverX siRNA Transfection Reagent 20 C DeliverX siRNA Buffer 1 RT DeliverX siRNA Buffer 2 RT DeliverX Plus siRNA Transfection Kit Contents Component Storage DeliverX Plus siRNA Transfection Reagent 20 C DeliverX Plus siRNA Buffer 1 RT DeliverX Plus siRNA Buffer 2 RT Divide transfection reagent into 50 100 uL aliquots to minimize freeze thaw
10. ed in the water during sonication DeliverX or DeliverX Plus SiRNA Transfection Sample Reagent uL Buffer 2 uL Tube 1 Target siRNA n Ae Tube 2 Negative 8 42 Control siRNA Tube 3 Cells only 20 a Recommended control not supplied with the kit Form concentrated siRNA siRNA transfection reagent complex a Match tubes with the same tube numbers from Step 2 and Step 3 b Mix the siRNA Buffer 1 solution 50 uL from Step 2 with the siRNA Transfection Reagent Buffer 2 solution 50 uL from Step 3 c Vortex for 3 seconds at speed 7 or pipet up and down several times d Incubate tubes at 37 C for 20 minutes Prepare complex dilution buffer by mixing equal volumes of Buffer 1 and Buffer 2 For example mix 500 uL of Buffer 1 and 500 uL of Buffer 2 to make 1 000 uL of complex dilution buffer Prepare working siRNA siRNA transfection reagent complexes by adding 300 uL of complex dilution buffer to the concentrated complex prepared in Step 4 The volume of each tube should be 400 uL Use this working complex for transfection or serially dilute it with the complex dilution buffer before the transfection DeliverX and DeliverX Plus Kit User Manual Page 11 Transfecting Adherent Cells Transfecting Adherent Cells Assay Preparation Pre warm both serum free and complete growth media to 37 C Procedure When performing transfections in different sized wells scale the amount of
11. iRNA control MLS Assay Workflow and Recommended Guidelines Controls and Optimization Assay Workflow Transfection Guidelines 4 Time Required 1 Prepare Adherent Cells Variable typically 16 24 hr a Plate cells before transfection 2 Prepare Transfection Complex 30 min a Prepare siRNA Buffer 1 b Prepare siRNA Transfection Reagent Buffer 2 c Combine siRNA Buffer 1 and siRNA Transfection Reagent Buffer 2 d Incubate complex at 37 C for 20 min 3 Transfect Cells Knockdown Target 24 72 hr a Add complex incubate 3 5 min at RT b Add serum free media incubate 2 4 hr at 37 C 596 CO c Add complete media incubate 24 72 hr at 37 C 5 CO 4 Quantify mRNA and or Protein Knockdown Variable depending on assay Use healthy cells in mid log phase not overgrown Determine optimal transfection conditions and use cells within 4 20 passages Passage number is cell type specific Do not use antibiotics during transfection process You can use antibiotics to maintain the cell line Use high quality high potency siRNAs Sonicate the transfection reagent as stated in the procedure to ensure proper formation of the siRNA siRNA transfection reagent complexes Note Once the siRNA siRNA transfection reagent complex has been formed properly it can be diluted to obtain varying siRNA concentrations Perform all procedures in a laminar flow hood using proper tissue culture techniques Run tripl
12. icates for each data point DeliverX and DeliverX Plus Kit User Manual Page 7 Assay Workflow and Recommended Guidelines Controls and Optimization Recommended Controls Optimizing Transfections Page 8 Proficiency Controls For new users or when working with a new cell type we recommend the use of the following proficiency controls FAM labeled siRNA delivery control This is a fluorescently labeled control for visualizing siRNA transfection efficiency under a fluorescent microscope Positive control siRNA Use a validated high potency siRNA as a functional SiRNA control Robust knockdown of that target ensures that the assay conditions are optimal for the cell type Routine Controls These controls assess the transfection and RNA knockdown efficiency and we recommend they be used routinely Negative control siRNA This control is typically an siRNA designed to have no homology to specified species and therefore no effect on mRNA or protein levels This control enables you to quantitate the amount of the mRNA knockdown Negative siRNA controls are also referred to as non silencing or scrambled SiRNAs Positive control siRNA This is a validated high potency siRNA whose functional response ensures that the assay is working in a reproducible manner Cells only This is a buffer only control serving as a mock transfection designed to monitor for any non transfection related phenomenon during the experiment Thi
13. ith minimal cell damage and good reproducibility when following the optimization guidelines provided in this User Manual Panomics DeliverX Plus siRNA Transfection Kit is a validated solution for many cell types Our DeliverX Plus siRNA Transfection Kits contain reagents particularly well suited for transfection of classically difficult to transfect cell types and comes with a certificate of analysis for each lot and validated protocols for a number of cell types There are currently 18 validated cell type specific protocols with additional protocols being introduced on a monthly basis The validated cell types protocols are 3T3 L1 differentiated mouse adipocytes HepG2 C2C12 undifferentiated mouse myocytes C2C12 differentiated mouse myotubes U87MG HUVEC BSMC Astrocytoma human cells NHEK AD MDA MB 231 HT29 RAW 264 7 A549 SW620 THP 1 A2780 ASPC 1 and MCF 7 Note Visit our website at www panomics com for the most up to date list of validated cell specific protocols available with our DeliverX Plus kits Regardless of which solution suits your needs each of these reagents offers unrivalled efficiency minimal toxicity and a simple workflow DeliverX Transfection Mechanism DeliverX transfection reagents are based on the novel delivery technology called MPG This technology was developed at Centre de Recherches en Biochimie Macromol culaire CNRS in Monpellier France in the laboratory of Dr F Heitz and Dr G Divit
14. llowing table provides guidelines for seeding different sized culture vessels to obtain 30 70 confluence after 24 hours of growth IMPORTANT Use cells from passages 4 20 cell type specific Optimal cell density is very important for obtaining the best results Note These numbers are approximate because the exact number of cells required depends on cell type size and growth rate If you are using a Then seed cells at a density of In a volume of 6 well plate 150 300 x 103 cells well 2 mL well 12 well plate 50 200 x 103 cells well 1 mL well 24 well plate 25 75 x 10 cells well 500 uL well 96 well plate 5 10 x 10 cells well 100 uL well Grow cells to 40 8096 confluence or to mid log phase The following table provides guidelines for the number of cells required for running transfections in different sized culture vessels IMPORTANT Optimal cell density is very important for obtaining the best results Note These numbers are approximate because the exact number of cells required depends on cell type size and growth rate If you are using a Then resuspend cells at a density of In a volume of 6 well plate 0 3 0 9 x 108 cells mL 1 mL well 12 well plate 0 1 0 4 x 106 cells mL 1 mL well 24 well plate 5 x 106 cells mL 30 uL well 96 well plate 1 x 106 cells mL 10 uL well DeliverX and DeliverX Plus Kit User Manual Page 9 Preparing the siRNA siR
15. low the recommended guidelines stated in the manual for addition of working transfection complex DeliverX and DeliverX Plus Kit User Manual Contacting Panomics Contacting Panomics Technical Help For technical questions contact our technical support group by telephone at 866 317 2626 or by email at techsupport panomics com or visit our website www panomics com for an updated list of FAQs and product support literature For Additional For information about Panomics products or for ordering information contact your Services Regional Sales Manager or visit our website at www panomics com DeliverX and DeliverX Plus Kit User Manual Page 17
16. ng Adherent Cells 0 0 0 ccs 12 Assay Preparation 2 fc de ecu Gk oS a 12 Procedure enai ses Reden eu qve ecient ee BA 12 Transfecting Cells in Suspension llle 13 About Transfecting Cells in Suspension illie 13 Assay Preparation eeri a eiaa nenn hh hn 13 Procedure ind asx iro Sarge ax Eo an a btt Tao 2090 ls pes Eae E De 13 Scaling Transfections 02 see EER AUT E EROAN AEE KEERA 14 About Preparing Bulk siRNA siRNA Transfection Reagent Complexes 14 Procedure 2 ee Slt esie er e Ee Wee es 14 Transfection Guidelines for Different Well Sizes llli 15 Adherent Cells toc a 2 34 N Easter Bade Delta SE AVES 15 Cells in Suspension ooo 15 Troubleshooting sid 0 2 ec ee 16 Problems and Recommended Actions 0000 cece eee eae 16 Contacting Panomics 2 in sea eder a pe ea ea a Ae eae ales ea 17 Technical Help suitable it att 17 For Additional Services ps siii a oana a an a ooo 17 DeliverX and DeliverX Plus Kit User Manual Page iii DeliverX and DeliverX Plus siRNA Transfection Kit and Manual Overview DeliverX and DeliverX Plus siRNA Transfection Kit and Manual Overview About the Kits About the Manual DeliverX siRNA reagents are available in two formats Our DeliverX siRNA Transfection Kit is suitable for transfection of most cell types DeliverX siRNA Transfection Kits contain the reagents required to efficiently transfect siRNAs into most cell types w
17. plexes Wells of a Wells of a Wells of a Wells of a Required Required 96 Well Plate 24 Well Plate 12 Well Plate 6 Well Plate 1 1 2mL 40 16 8 4 2 2 4 mL 80 32 16 8 3 3 6 mL 120 48 32 12 4 4 8 mL 160 64 48 16 5 6 0 mL 200 80 64 20 Scaling up working siRNA siRNA transfection reagent complexes Working siRNA Complex Transfection 5 uM siRNA Transfection Dilution Complex Scale uL Buffer 1 uL Reagent uL Buffer 2 uL Buffer uL Formed mL 1X 13 37 8 42 300 0 4 2X 26 74 16 84 600 0 8 3x 39 111 24 126 900 1 2 Page 14 DeliverX and DeliverX Plus Kit User Manual Transfection Guidelines for Different Well Sizes Transfection Guidelines for Different Well Sizes Adherent Cells The tables below are a guideline based on surface areas for scaling the components of transfection for different well sizes Preparing transfections of adherent cells using various sizes of wells Working siRNA If you are using Using PBS Wash Transfection Complete Growth a cells with Complex Serum Free Media Media 6 well plate 500 uL 300 uL 300 uL 1 000 uL 12 well plate 300 uL 150 uL 150 uL 500 uL 24 well plate 150 uL 75 uL 75 uL 250 uL 96 well plate 100 uL 30 uL 30 uL 100 uL Cells in Suspension Preparing transfections of cells in suspension using various sizes of wells Resuspend Cell Working siRNA If you are Cells with PBS Suspension Transfection Serum Free Complete using a to
18. s 5 Pellet the cells at 400 x g for 5 minutes 6 Gently aspirate the PBS avoiding cell loss and resuspend with 300 uL of working siRNA siRNA transfection reagent complex as described in Preparing the siRNA siRNA Transfection Reagent Complex on page 10 7 Mix by pipetting up and down several times 8 Incubate at room temperature for 3 5 minutes 9 Add 300 uL of serum free media to the tube and mix gently by pipetting up and down several times 10 Transfer 600 uL of cells transfection complex to each well of a 6 well plate and incubate under normal cell culture conditions typically 37 C and 5 COs for 2 4 hours Note For maximum knockdown we recommend 4 hours 11 Add 1 mL well of complete growth media Note At this point the concentration of siRNA in the sample well is 30 nM 12 Incubate samples under normal cell culture conditions for 24 hours or the desired time interval Note 24 48 hour incubation times are typical when measuring MRNA knockdown 48 72 hours incubation times are typical when measuring protein knockdown For longer incubation times you may need to change the media 24 hours post transfection 13 Measure mRNA and or protein knockdown as appropriate We recommend quantitating mRNA expression levels with the Quantigene Reagent System DeliverX and DeliverX Plus Kit User Manual Page 13 Scaling Transfections Scaling Transfections Because the complex formation i
19. s control contains the transfection buffers but does not include either the transfection reagent or siRNA Note DeliverX or DeliverX Plus siRNA transfection reagent alone should not be used as a negative control as it is known to result in increased cytotoxicity Because cell types can differ significantly with respect to their capacity to be transfected we recommend that you optimize the protocol empirically The most important parameters for optimization are cell density and siRNA concentration Cell Density Cell densities that are too high or too low may not take up an optimal amount of the complexes resulting in insufficient gene silencing or elevated levels of cytotoxicity Lower cell densities may be necessary for post transfection incubation times greater than 48 hours siRNA Concentration The siRNAs capacity for gene silencing are influenced in part by the siRNA design and properties of the target gene This may include mRNA localization stability and abundance and protein stability and abundance If too much siRNA is used during transfection you may see toxicity If too little is used you may not see adequate knockdown DeliverX and DeliverX Plus Kit User Manual Preparing the Cells the Day Before Transfection Preparing the Cells the Day Before Transfection Adherent Cell Types Suspension Cells or Adherent Cells in Suspension For most adherent cell types the optimal confluency for transfection is 30 70 The fo
20. s impaired when large volumes are prepared we recommend scaling up the working siRNA siRNA transfection reagent complex in 3X volume aliquots when preparing to transfect large sample numbers About Preparing Bulk siRNA siRNA Transfection Reagent Multiple replicate working transfection complexes should be prepared in parallel and Complexes nen combined into a single tube to produce bulk volume of working transfection complexes Procedure To scale up working siRNA transfection complexes Step Action 1 Determine the volume of working siRNA siRNA transfection reagent complex required for your culture vessel size and assay design 2 Use the table below to determine the number of 3X working transfection complexes required 3 Prepare the required number of working siRNA siRNA transfection reagent complexes in parallel following the procedure on page 10 and using the volumes provided in the table below 4 Combine the prepared working siRNA siRNA transfection reagent complexes to form a single bulk solution 5 Use this preparation for transfection or if required serially dilute with the complex dilution buffer before the transfection Preparing multiple replicate 3X working siRNA siRNA transfection reagent complexes in parallel for generating bulk volumes Number of 3X Combined Bulk Working Working Transfection Transfection Complexes Com
21. t the time of transfection Transfection time not adequate Evaluate transfection times greater than 4 hours Sub optimal post transfection incubation time Test a range of 24 72 hours for post transfection incubation time Cell have a tendency to grow in groups or clumps Do not tap flask during trypsinization Allow the cells to detach themselves After trypsinizing the cells pipet up and down several times to release the cells Seed them at the desired density Visually check by microscopy Cell response changes after repeated passages Thaw fresh cells for subsequent experiments Avoid using cells at early or late passages Medium depletion For longer incubation times 48 72 hours you may have to change the medium 24 hours post transfection Alternatively add additional complete medium at 24 hours High toxicity DeliverX or DeliverX Plus siRNA transfection reagent siRNA complex not formed properly Do not exceed the recommended volume of transfection reagent when preparing the transfection reagent complex Cell density too low Evaluate higher cell densities at the time of transfection Be gentle when removing medium or PBS during washes Cells become more sensitive to reagents after repeated passages Thaw fresh cells for subsequent experiments Avoid using cells at early or late passages Too much working transfection complex added to the cells Fol
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