User Manual - Biomol GmbH
Contents
1. Cignal Cignal Cignal Small Opti MEM SureFECT Opti MEM Time of Reporter Negative Positive Molecule DNA diluent per well SureFECT Transfection per well Control Control Organic per well diluent hours per well Construct Compound per well per well per well 1 100 ng 25 ul 0 3 pl 25 ul 1 0 ul i H H 100 ng a 2 1 0 ul 1X 25 ul 0 3 ul 25 ul 100 ng 3 1 0 ul 10X 25 ul 0 3 ul 25 ul 100 ng 4 1 0 ul 100X 25 ul 0 3 ul 25 ul 100 ng 5 1 0 ul 25 ul 0 3 ul 25 ul 30 hor 42h 100 ng 6 1 0 ul 1X 25 ul 0 3 pl 25 ul 100 ng 7 1 0 ul 10X 25 ul 0 3 ul 25 ul 100 ng 8 1 0 ul 100X 25 ul 0 3 ul 25 ul 100 ng 9 1 0 ul 25 ul 0 3 ul 25 ul 31X is a smallest appropriate amount of small molecule or organic compound expected to modulate signaling pathway 1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 Technical Support support SABiosciences com www SABiosciences com 20 Version 1 5 Set up three polystyrene tubes as follows Experimental transfections Tubes 1 4 100 pl Opti ME2. Product Name Catalog Number Cignal Finder Cancer 10 Pathway Reporter Array CCA 001L Cignal Finder Immune Response 10 Pathway Reporter Array CCA 002L Cignal Finder Development 10 Pathway Reporter Array CCA 003L Cignal Finder Toxicity 10 Pathway Reporter Array CCA 004L Technical Support support SABiosciences com www SABiosciences com 4 Version 1 5 12 Tube Strip CAAA Add SureFECT Transfection Reagent Cignal Reporter amp Test Nucleic Acids Treat amp Analyze Phenotype with a Cell based Assay Oual luciferase Figure 1 Overview of Cignal Finder Reporter Array tube format Process Technical Support 888 503 3187 US 301 682 9200 5 Cignal Finder Reporter Arrays tube format ll Product Contents and Descriptions A Cignal Finder 10 Pathway Reporter Array Contents Table 1 Cignal Finder Reporter Array tube format Specifications Concentration Component Specification and total volume A mixture of an inducible transcription factor Each of the responsive firefly luciferase reporter and 100 ng ul 100 pl 10 Reporter constitutively expressing Renilla construct Assays 40 1 Negative A mixture of non inducible firefly luciferase control reporter and constitutively expressing 100 ng ul 100 ul Renilla construct 40 1 A mixture of a constitutively expressing GFP Positive construct constitutively expressing firefly 100 ng ul 100 ul co
3. 3 After the 5 minute incubation add 25 ul of diluted SureFECT into each of the five tubes containing 25 ul of diluted constructs 1 1 ratio as detailed in Table 3 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in a culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 CO2 Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately measure the cell density with a hemacytometer or automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specific constructs shnRNA SureFECT complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing construct shRNA SureFECT complexes This gives a final volume of 150 ul Mix gently by rocking the plate back and forth 8 Incubate cells at 37 C in a 5 CO incubator for 16 24 hours 9 After 16 24 hours of transfection change the medium to complete growth medium DMEM with 10 FBS 0 1m
4. Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support 888 503 3187 US 301 682 9200 25 Cignal Finder Reporter Arrays tube format H Scaling up transfection experiments To transfect cells in different tissue culture formats vary the amounts of constructs number of cells and volume of SureFECT and medium used in proportion to the surface area aS shown in the Table 7 The parameters shown in Table 7 are standardized for HEK 293H cells Use these parameters as a starting point to optimize transfections for your cell line of interest Table 7 Reagent amounts for transfecting cells in different size culture vessels Type of Surface Starting Starting Volume of Starting Volume of siRNA shRNA Plate Area amount of Volume of Cell No of Opti MEM Vector or Gene cm per construct SureFECT Suspension Adherent Medium Expression Vector well ng well ul well ul well Cells per ul per Well Well 96 well 0 3 100 0 3 100 20 000 2 X 25 2 pmol 200 ng 48 well 0 95 150 0 8 250 62 500 2X50 5 pmol 500 ng 24 well 1 9 250 1 6 500 125 000 2X50 10 pmol 750 ng 12 well 3 8 500 3 2 1000 250 000 2 X 100 20 pmol 1 5 ug 6 well 9 4 1000 8 0 2500 750 000 2 X 250 50 pmol 4 0 ug 35 mm 8 0 1000 8 0 2500 750 000 2 X 250 50 pmol 4 0 ug 6 60mm 21 2000 18 0 5000 1 5X 10 2 X 500 100 pmol 8 0 ug 6 100mm 55 5000 45 0 15000 15ml 4 5X 10
5. in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 25 ul of diluted SureFECT into each of the five tubes containing 25 ul of the diluted nucleic acids 1 1 ratio as detailed in Table 2 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in a culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 CO2 Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately measure the cell density with a hemacytometer or an automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specific constructs sIRNA SureFECT complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing constructs siIRNA SureFECT complexes This gives a final volume of 150 ul Mix gently by rocking the plate back and forth 8 Incubate cells at 37 C in a 5 CO incubator for 16 24 hours 9
6. 3 1 0 ul 2 pmol 25 ul 0 3 ul 25 ul 48 hor 72h 100 ng 4 1 0 ul 2 pmol 25 ul 0 3 ul 25 ul 100 ng 5 1 0 ul 25 ul 0 3 ul 25 ul 1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 Add 25 ul of Opti MEM to each of 5 polystyrene tubes avoid using DMEM along with the following Experimental transfection 1 ul 100 ng Cignal reporter 2 pmol sequence specific siRNA Control transfections 1 ul 100 ng Cignal reporter 2 pmol negative control siRNA 1 ul 100 ng Cignal negative control 2 pmol sequence specific siRNA 1 ul 100 ng Cignal negative control 2 pmol negative control siRNA 1 ul 100 ng Cignal positive control Mix each transfection cocktail gently Technical Support 888 503 3187 US 301 682 9200 13 Cignal Finder Reporter Arrays tube format 2 Prepare a SureFECT dilution for 5 tubes mentioned in step 1 by dispensing 1 5 ul of SureFECT into 125 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium
7. Add SureFECT Transfection me Test Nucleic Acids DAY 2 DAY 1 Trypsinize if necessary count and suspend cells to appropriate density Seed cells into multiwell plate s DAY 2 Prepare nucleic acid mixtures in appropriate ratios This may include any of the following combinations depending upon the experimental design we recommend carrying out each transfection condition in triplicate Experimental transfection i Cignal Reporter test nucleic acid expression plasmids shRNA plasmids or siRNAs Control transfections ii Cignal Reporter negative control for test nucleic acid iii Cignal Negative Control test nucleic acid expression plasmids shRNA plasmids or siRNAs iv Cignal Negative Control negative control for test nucleic acid v Cignal Positive Control Dilute transfection reagent into appropriate medium If you are using a transfection reagent other than SureFECT follow their manufacturer s protocol for transfection Add diluted transfection reagent to nucleic acid mixtures incubate at room temperature for 20 minutes Aliquot transfection complexes into wells containing overnight cell cultures Technical Support support SABiosciences com www SABiosciences com 12 Version 1 5 C Co transfection Protocol for siRNA Reporter Assay The following protocol is designed to reverse transfect an adherent cell line HEK 293H using SureFECT Transfection Reagent Cat No SA 01 in a 96 well plate
8. Nucleic Acids 9090 Seed Cells for Reverse Transfection OC DAY 1 LEE DAY 1 Prepare nucleic acid mixtures in appropriate ratios This may include any of the following combinations depending upon the experimental design we recommend carrying out each transfection condition in triplicate Experimental transfection i Cignal Reporter test nucleic acid expression plasmids shRNA plasmids or siRNAs Control transfections ii Cignal Reporter negative control for test nucleic acid iii Cignal Negative Control test nucleic acid expression plasmids shRNA plasmids or siRNAs 7 Cignal Negative Control negative control for test nucleic acid Cignal Positive Control n Dilute SureFECT into Opti MEM Add diluted SureFECT to nucleic acid mixtures incubate at room temperature for 20 minutes Trypsinize if necessary count and suspend cells to appropriate density Aliquot transfection complexes into wells Immediately seed cells to each well Technical Support 888 503 3187 US 301 682 9200 11 Cignal Finder Reporter Arrays tube format For detailed information on the transfection conditions and treatment of cultures post transfection refer to the application specific protocols within this user manual 2 Traditional Transfection Protocol Overview 2 DAY PROCEDURE Traditional Transfection Add Cells and Medium 009 ac 96 well Cell Culture Plate LEE DAY 1 Cignal Reporter and
9. control of a basal promoter element TATA box joined to tandem repeats of a specific Transcriptional Response Element TRE Figure 2A This construct monitors both increases and decreases in the activity of a key transcription factor which is a downstream target of a specific signaling pathway The constitutively expressing Renilla construct encodes the Renilla luciferase reporter gene under the control of a CMV immediate early enhancer promoter Figure 2B and acts as an internal control for normalizing transfection efficiencies and monitoring cell viability It is also useful to confirm transfection and to verify active luciferase in the transfected culture 2 Negative control The negative control is a mixture of non inducible reporter construct and constitutively expressing Renilla luciferase construct 40 1 The non inducible reporter construct encodes firefly luciferase under the control of a basal promoter element TATA box without any additional transcriptional response elements Figure 2C The negative control is critical to identifying specific effects and determining background reporter activity 3 Positive control The positive control is a constitutively expressing GFP construct Figure 2D pre mixed with a constitutively expressing firefly luciferase construct Figure 2E and a constitutively expressing Renilla luciferase construct Figure 2B 40 1 1 The positive control is necessary for visual confirmation of transfection
10. format The Cignal Reporter Assays work well with transfection reagents from other vendors f you are using a transfection reagent other than SureFECT follow their manufacturer s protocol for optimizing transfection The Cignal Reporter Assays also work well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the components in proportion to the surface area see section IV H This is just a general quideline the optimal conditions amounts should be optimized according to the cell type and the study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 2 Guidelines for setting up co transfections of siRNA and Cignal Reporter Assays Table 2 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol Cignal Cignal Cignal Specific Negative Opti MEM SureFECT Opti MEM Time of Reporter Negative Positive siRNA Control Nucleic per well SureFECT transfection per well Control Control per well siRNA Acid Diluent hours per well per well per well Diluent per well per well 100 ng 1 1 0 ul 2 pmol 25 ul 0 3 ul 25 ul 100 ng 2 1 0 ul 2 pmol 25 ul 0 3 ul 25 ul 100 ng
11. transfection protocols Moreover if you are using plates or wells of different size adjust the component in proportion to the surface area see section IV F This is just a general quideline the optimal conditions amounts should be adjusted according to the cell type and the study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 6 Guidelines for studying the effect of a peptide or recombinant protein Table 6 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol Cignal Cignal Cignal Peptide or Opti MEM SureFECT Opti MEM Time of Reporter Negative Positive Recombinant DNA Diluent per well SureFECT Transfection per well Control Control Protein per well diluent hours per well per well per well per well 1 ei 25 ul 0 3 ul 25 ul 2 es 0 1x 25 ul 0 3 pl 25 ul 3 ao a 10X 25 ul 0 3 ul 25 ul 4 ai k 100X 25 ul 0 3 ul 25 ul 5 Ho D 25 ul 0 3 ul 25 ul 30 h or 42h 6 X i 1X 25 ul 0 3 pl 25 ul 7 ad a 10X 25 ul 0 3 ul 25 ul 8 ae a 100X 25 ul 0 3 ul 25 ul 9 AG i 25 ul 0 3 ul 25 ul 4X is a smallest appropriate amount of interfering peptide recombinant protein growth factor
12. 0 3 ul 25 ul 100 ng 5 1 0 ul 25 ul 0 3 ul 25 ul 1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 Add 25 ul of Opti MEM to each of 5 polystyrene tubes avoid using DMEM along with the following Experimental transfection 1 ul 100 ng Cignal reporter 200 ng sequence specific snRNA Control transfections 1 ul 100 ng Cignal reporter 200 ng negative control shRNA 1 ul 100 ng Cignal negative control 200 ng sequence specific snRNA 1 ul 100 ng Cignal negative control 200 ng negative control shRNA 1 ul 100 ng Cignal positive control Mix each transfection cocktail gently Technical Support 888 503 3187 US 301 682 9200 15 Cignal Finder Reporter Arrays tube format 2 Prepare a SureFECT dilution for 5 tubes mentioned in step 1 by dispensing 1 5 ul of SureFECT into 125 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes
13. 2 X 1500 300 pmol 25 pg a 2X means one volume of Opti MEM medium for diluting constructs and another volume of Opti MEM medium for diluting SureFECT For any other troubleshooting or technical questions about the Cignal Reporter Assay please call one of our Technical Support representatives at 1 888 503 3187 or 301 682 9200 or email at support SABiosciences com Technical Support support SABiosciences com 26 www SABiosciences com Appendix Cignal Finder 10 Pathway Reporter Arrays Cignal Finder Cancer 10 Pathway Reporter Array Tube Format CCA 001L Plate Format CCA 101L Version 1 5 Transcriptional Regulatory Element TRE Pathway Transcription Factor Wnt TCF LEF response element TCF LEF Notch RBP Jx binding element RBP J p53 DNA Damage p53 response element p53 TGFB SMAD response element SMAD2 SMAD3 SMAD4 Cell Cycle pRb E2F E2F binding element E2F DP1 NFkB NF B binding element NFkB Myc Max E box binding element Myc Max Hypoxia HIF response element Hypoxia inducible factor 1 HIF 1 MAPK ERK Serum response element SRE Elk 1 SRF MAPK JNK AP 1 binding element AP 1 Cignal Finder Immune Response 10 Pathway Reporter Array Tube Format CCA 002L Plate Format CCA 102L Transcriptional Regulatory Element TRE Pathway Transcription Factor NFkB NF B binding element NF B PKC Ca NFAT response element NFAT Type Int
14. 5 CO2 Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately determine the cell density with a hemacytometer or an automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specific complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing constructs SureFECT complexes This gives a final volume of 150 ul Mix gently by rocking the plate back and forth 8 Incubate cells at 37 C in a 5 CQz incubator for 16 hours 9 After 16 hours of transfection change medium to assay medium Opti MEM containing 0 5 of fetal bovine serum 1 NEAA 100 U ml Penicillin and 100 ug ml Streptomycin Technical Support 888 503 3187 US 301 682 9200 21 Cignal Finder Reporter Arrays tube format 10 After 24 hours of transfection treat the cells as described in Table 5 with 1X 10X and 100X amount of small molecule or organic compound 1X is the lowest appropriate amount of small molecule or organic compound expected to modulate the signaling pathway 11 To study the effect of small molecule or organic compound we re
15. After 16 24 hours of transfection change the medium to complete growth medium DMEM with 10 FBS 0 1mM NEAA 1mM Sodium pyruvate 100 U ml penicillin and 100 ug ml streptomycin 10 To study the effect of knockdown we recommend harvesting cells 48 or 72 hours after transfection to perform dual luciferase assay 12 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay We have found that some components in DMEM interfere with SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that have been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support support SABiosciences com www SABiosciences com 14 Version 1 5 D Co transfection Protocol for shRNA Reporter Assay The following protocol is designed to reverse transfect an adherent cell line HEK 293H using SureFECT Transfection Reagent Cat No SA 01 in a 96 well plate format The Cignal Reporter Assays work well with transfection reagents from o
16. Cignal positive control reporters which are each under the control of the CMV enhancer promoter cassette If your stimulus is one of the very few reported activators of the CMV regulatory element we advise using an alternative reporter as an internal control e W Bruening B Giasson W Mushynski and H D Durham 1998 Nucleic Acids Research 26 2 486 489 Activation of stress activated MAP protein kinases up regulates expression of transgenes driven by the cytomegalovirus immediate early promoter e Madhu S Malo Moushumi Mozumder Alexander Chen Golam Mostafa Xiao Bo Zhang Richard A Hodin 2006 Analytical Biochemistry 350 307 309 pFRL7 An ideal vector for eukaryotic promoter analysis Technical Support support SABiosciences com www SABiosciences com 8 Version 1 5 Additional Materials Required Mammalian cell line cultured in the appropriate growth medium Cell culture medium and standard cell culture supplies 96 well tissue culture plates Multi channel pipettor and pipettor reservoirs Transfection reagent We recommend SureFECT SABiosciences Cat No SA 01 however other transfection reagents work equally well Polystyrene test tubes BD FALCON Cat 352099 Opti MEM Reduced Serum Medium Invitrogen Cat No 31985 062 Fetal bovine serum FBS Non essential amino acids NEAA Invitrogen Cat No 11140 050 Penicillin Streptomycin Hemacytometer Dual Luciferase Assay System o Dual Luciferase Rep
17. Cignal Finder Reporter Arrays are valuable tools for progressing from the identification of genes proteins or small molecules to understanding their function Each pathway focused dual luciferase reporter encodes for the mammalian codon optimized non secreted form of the firefly luciferase gene carrying a protein destabilizing sequence Cells rapidly degrade the destabilized form of the firefly luciferase protein and hence the background luciferase activity noise level is greatly reduced Due to low background activity the magnitude of the response that can be measured signal to noise ratio as well as the speed of measuring changes in transcription are enhanced The Cignal dual luciferase reporter assays provide outstanding reproducibility sensitivity specificity and signal to noise ratio They are extremely useful assays for carrying out quantitative pathway regulation studies Benefits of Cignal Finder 10 Pathway Reporter Arrays e BIOLOGICAL PROCESS FOCUSED Profile the changes in the activities of ten signaling pathways relevant to a specific biological process e HIGH PERFORMANCE Dual luciferase assay provides high sensitivity specificity and reproducibility e FLEXIBILITY AND CONVENIENCE Utilize a straightforward traditional transfection or reverse transfection procedure with your favorite cell lines to rapidly generate valuable mechanism of action data Available Cignal Finder 10 Pathway Reporter Arrays tube format
18. It is also useful for transfection optimization studies The expression of the GFP from the positive control construct can be monitored by fluorescence microscopy using an excitation filter of 470 20 nm 470 40 nm and an emission filter of 515 nm long pass Technical Support 888 503 3187 US 301 682 9200 7 Cignal Finder Reporter Arrays tube format Tandem repeats TATA box of TREs A Firefly Luc B n CMV immediate early enhancer promoter TATA box y C Firefly Luc D 1 enhancer promoter CMV immediate early enhancer promoter Firefly Luc Figure 2 Schematic representation of constructs involved in the Cignal Reporter Assay A The inducible transcription factor responsive construct expressing firefly luciferase B The constitutively expressing Renilla luciferase construct C The non inducible firefly luciferase reporter construct D The constitutively expressing GFP construct and E The constitutively expressing firefly luciferase construct IMPORTANT NOTE There are a few reports in the literature of the CMV regulatory element being activated by certain stimuli see below We recommend that you confirm that the stimulus used in each Cignal reporter assay does not induce the CMV regulatory element in order to confirm that the CMV Renilla construct is the appropriate normalization construct for your experiment This can be done empirically by testing the impact of your stimulus on the
19. J SABiosciences Cea zens CY biomol www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D User Manual Cignal Finder 10 Pathway Reporter Arrays tube format Cell Based Multi Pathway Activity Assays See Purchaser Notification for limited use license and warranty information pages 2 and 3 Part 1034A Version 1 4 8 18 2008 SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA Cignal Finder 10 Pathway Reporter Arrays tube format Cell Based Multi Pathway Activity Assays User Manual BIOMOL GmbH For Catalog Numbers CCA 0XXL se fons O biomol A meme or 0800 2466651 D Ordering and Technical Service Contact Information Fax 49 40 85326022 or 0800 2466652 D e Tel 1 888 503 3187 US 301 682 9200 outside US e Fax 1 888 465 9859 US 301 682 7300 outside US e On line Order www SABiosciences com e E MAIL order SABiosciences com to place an order support SABiosciences com for technical support You may place orders by fax e mail or from our website Each order should include the following information Your contact information name phone email address Product name catalog number and quantity Purchase order number or credit card information Visa or MasterCard Shipping address Billing address For more information visit us at www SABiosciences com LIMITED PRODUCT WARRANTY This product is intended for research purposes only
20. M 4 ul 400 ng Cignal reporter 4 volumes for conditions 1 to 4 of Table 5 for every well dilute 1 ul 100 ng of Cignal reporter in 25 ul of Opti MEM serum free culture medium Control transfections Tubes 5 8 100 ul Opti MEM 4 ul 400 ng Cignal negative control 4 volumes for conditions 5 to 8 of Table 5 for every well dilute 1 ul 100 ng of Cignal reporter in 25 ul of Opti MEM serum free culture medium Tube 9 25 ul Opti MEM 1ul 100 ng Cignal positive control Mix each transfection cocktail gently 2 Prepare a SureFECT dilution for 9 tubes mentioned in step 1 by dispensing 2 7 ul of SureFECT into 225 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 100 ul of diluted SureFECT to the two tubes mentioned in step 1 containing equal volume 100 ul of diluted Cignal reporter and add 25 ul of diluted SureFECT into the positive control tube containing 25 ul of diluted constructs 1 1 ratio as detailed in Table 5 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing
21. M NEAA 1mM Sodium pyruvate 100 U ml penicillin and 100 ug ml streptomycin 10 To study the effect of knockdown we recommend harvesting cells 48 or 72 hours after transfection to perform dual luciferase assay 11 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay We have found that some components in DMEM interfere with SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that have been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support support SABiosciences com www SABiosciences com 16 Version 1 5 E Co transfection Protocol for Expression Vector Reporter Assay The following protocol is designed to reverse transfect an adherent cell line HEK 293H using SureFECT Transfection Reagent Cat No SA 01 in a 96 well plate format The Cignal Reporter Assays work well with transfection reagents from other vendors f you are using transfection reagent other than SureFECT follow their manu
22. Use low passage cells that are actively growing and are greater than 90 viable for maximal transfection efficiencies D Do not add antibiotics to media during transfection as this may cause cell death Take care to always seed the same number of cells in each well in order to maximize the reproducibility of your experiment F Serum induces various signaling pathways leading to cross talk and high background Therefore use reduced amounts of serum 0 5 in the assay medium during the experimental treatment to minimize these serum effects m Technical Support support SABiosciences com www SABiosciences com 10 Version 1 5 B Generalized Transfection Protocols We recommend using reverse transfection protocols with the SureFECT transfection reagent throughout the Cignal Finder Reporter Arrays User Manual This is due to the time savings and improved reproducibility of using this method compared to traditional forward transfection methods However the Cignal Reporter Assays included in each Cignal Finder Array also work well with traditional forward transfection methods and transfection reagents from other vendors Below are general protocol overviews for the Cignal Reporter Assays using either reverse or forward transfection approaches 1 Reverse Transfection Protocol Overview 1 DAY PROCEDURE Reverse Transfection 96 well Cell Culture Plate LEZLEY Add SureFECT Transfection Reagent Cignal Reporter and Test
23. and is not intended for drug or diagnostic purposes or for human use This warranty limits our liability to replace this product in the event the product fails to perform due to any manufacturing defect SABiosciences Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SABiosciences Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product NOTICE TO PURCHASER The purchase of Cignal Finder Reporter Arrays includes a limited nonexclusive license to use the kit components for research use only This license does not grant rights to use the kit components for reproduction of any constructs to modify kit components for resale or to manufacture commercial products without written approval of SABiosciences Corporation No other license expressed implied or by estoppel is granted NOTICE TO PURCHASER II The Dual Luciferase Reporter Assay is a trademark of Promega Corporation SABiosciences Corporation 6951 Executive Way Suite 100 Frederick MD 21703 USA CONTENTS l Introduction 4 II Product Contents and Descriptions 6 III Additional Materials Required 9 IV Protocol 10 A Before you begin 10 B Generalized Transfection Protocols 11 C Co transfection Protocol for siRNA Reporter Assay 13 D Co transfect
24. asmid 1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 888 503 3187 US 17 Technical Support 301 682 9200 Cignal Finder Reporter Arrays tube format Add 25 ul of Opti MEM to each of 9 polystyrene tubes avoid using DMEM along with the following Experimental transfections 1 ul 100 ng Cignal reporter 100 ng experimental vector expressing gene of interest 100 ng carrier DNA 1 ul 100 ng Cignal reporter 200 ng experimental vector expressing gene of interest Control transfections 1 ul 100 ng Cignal reporter 100 ng negative control expression vector 100 ng carrier DNA 1 ul 100 ng Cignal reporter 200 ng negative control expression vector 1 ul 100 ng Cignal negative control 100 ng experimental vector expressing gene of interest 100 ng carrier DNA 1 ul 100 ng Cignal negative control 200 ng experimental vector expressing gene of interest 1 ul 100 ng Cignal negative control 100 ng negative control expression vector 100 ng carrier DNA 1 ul 100 ng Cignal negative control 200 ng negat
25. commend harvesting cells 6 hours or 18 hours after treatment to perform dual luciferase assay 12 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay We found some components in DMEM interfere with the SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that had been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support support SABiosciences com www SABiosciences com 22 Version 1 5 G Transfection and Treatment Protocol for Reporter Assay Peptide Recombinant Protein The following protocol is designed to reverse transfect an adherent cell line HEK 293H using SureFECT Transfection Reagent Cat No SA 01 in a 96 well plate format The Cignal Reporter Assays work well with transfection reagents from other vendors f you are using transfection reagent other than SureFECT follow their manufacturer s protocol for transfection The Cignal Reporter Assays also work well using traditional forward
26. e medium in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 100 ul of diluted SureFECT to the two tubes mentioned in step 1 containing equal volume 100 ul of diluted Cignal reporter and add 25 ul of diluted SureFECT into the positive control tube containing 25 ul of diluted constructs 1 1 ratio as detailed in Table 6 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 CO2 Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately determine the cell density with a hemacytometer or an automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specific complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing constructs SureFECT complexes This gives a final volume of 150 ul Mix gently by roc
27. erferon Interferon stimulated response element ISRE STAT1 STAT2 Interferon Gamma Interferon gamma activation sequence GAS STAT1 STAT1 MAPK ERK Serum response element SRE Elk 1 SRF MAPK JNK AP 1 binding element AP 1 TGFB SMAD response element SMAD2 SMAD3 SMAD4 cAMP PKA cAMP regulatory element CRE CREB C EBP C EBP binding element C EBP Glucocorticoid Receptor Glucocorticoid response element GRE Glucocorticoid Receptor GR Technical Support 888 503 3187 US 27 301 682 9200 Cignal Finder Reporter Arrays tube format Cignal Finder 10 Pathway Reporter Arrays continued Cignal Finder Development 10 Pathway Reporter Array Tube Format CCA 003L Plate Format CCA 103L Transcriptional Regulatory Element TRE Pathway Transcription Factor Notch RBP J binding element RBP J Wnt TCF LEF response element TCF LEF Myc Max E box binding element Myc Max NF B NF B binding element NFkB TGFB SMAD response element SMAD2 SMAD3 SMAD4 Cell Cycle pRb E2F E2F binding element E2F DP1 C EBP C EBP binding element C EBP cAMP PKA cAMP regulatory element CRE CREB MAPK ERK Serum response element SRE Elk 1 SRF MAPK JNK AP 1 binding element AP 1 Cignal Finder Toxicity 10 Pathway Reporter Array Tube Format CCA 004L Plate Format CCA 104L Pathway Transcriptional Regulatory Element TRE Transcription Factor p53 DNA Damage p53 response ele
28. expected to modulate signaling pathway _1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 Technical Support 888 503 3187 US 301 682 9200 23 Cignal Finder Reporter Arrays tube format Set up three polystyrene tubes as follows Experimental transfections Tubes 1 4 100 pl Opti MEM 4 ul 400 ng Cignal reporter 4 volumes for conditions 1 to 4 of Table 6 for every well dilute 1 ul 100 ng of Cignal reporter in 25 ul of Opti MEM serum free culture medium Control transfections Tubes 5 8 100 ul Opti MEM 4 ul 400 ng Cignal negative control 4 volumes for conditions 5 to 8 of Table 6 for every well dilute 1 ul 100 ng of Cignal reporter in 25 ul of Opti MEM serum free culture medium Tube 9 25 ul Opti MEM 1ul 100 ng Cignal positive control Mix each transfection cocktail gently 2 Prepare a SureFECT dilution for 9 tubes mentioned in step 1 by dispensing 2 7 ul of SureFECT into 225 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free cultur
29. f prepared cell suspension 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing construct vector SureFECT complexes This gives a final volume of 150 ul Mix gently by rocking the plate back and forth Technical Support support SABiosciences com www SABiosciences com 18 Version 1 5 8 Incubate cells at 37 C in a 5 CO 2 incubator for 16 24 hours 9 After 16 24 hours of transfection change the medium to complete growth medium DMEM with 10 FBS 0 1mM NEAA 1mM Sodium pyruvate 100 U ml penicillin and 100 ug ml streptomycin 11 To study the effect of the gene product we recommend harvesting cells 32 hours or 48 hours after transfection to perform the dual luciferase assay 12 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay We have found that some components in DMEM interfere with SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that have been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells ot
30. facturer s protocol for transfection The Cignal Reporter Assays also work well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the component in proportion to the surface area see section IV H This is just a general quideline the optimal conditions amounts should be adjusted according to the cell type and the study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 4 Guidelines for setting up co transfections of an expression vector and Cignal Reporter Assay Table 4 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol 53s ee ee ee ficos Pos ae i ai EPE Re akgal gin Og O26 20a 8 376E 2 87552 O eea8 32 seas FES Wi W zZ 1 a ah 100 ng pa 25 ul 0 3 ul 25 ul 2 i D 200 ng 25 ul 0 3 ul 25 ul 3 es i 100 ng AN 25 ul 0 3 pl 25ul 32h 48h 4 a a 200 ng 25 ul 0 3 ul 25 ul 5 ne a 100 ng a 25 ul 0 3 ul 25 ul 6 a is 200 ng 25 ul 0 3 ul 25 ul 7 no i 100 ng ee 25 ul 0 3 ul 25 ul 8 l P ia 200 ng 25 ul 0 3 ul 25 ul 9 hie a 25 ul 0 3 ul 25 ul Carrier DNA means any empty plasmid such as a pUC or a pBR pl
31. herwise increase the cell numbers Technical Support 888 503 3187 US 301 682 9200 19 Cignal Finder Reporter Arrays tube format F Transfection and Treatment Protocol for Reporter Assay Small Molecules Organic Compounds The following protocol is designed to reverse transfect an adherent cell line HEK 293H using SureFECT SABiosciences Cat No SA 01 as a transfection reagent in 96 well plate format The Cignal Reporter Assays work well with transfection reagents from other vendors f you are using transfection reagent other than SureFECT follow their manufacturer s protocol for transfection The Cignal Reporter Assays also work well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the component in proportion to the surface area see section IV H This is just a general guideline the optimal conditions amounts should be adjusted according to the cell type and the study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 5 Guidelines for studying the effect of small molecules organic compounds Table 5 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol
32. ion Protocol for shRNA Reporter Assay 15 E Co transfection Protocol for Expression Vector Reporter Assay ue F Transfection and Treatment Protocol for Reporter Assay 20 Small Molecules Organic Compounds G Transfection and Treatment Protocol for Reporter Assay 23 Peptide Recombinant Protein H Scaling up Transfection Experiments 26 Appendix Cignal Finder 10 Pathway Array Product Descriptions 27 Technical Support support SABiosciences com www SABiosciences com 3 Cignal Finder Reporter Arrays tube format Introduction The Cignal Finder 10 Pathway Reporter Arrays enable you to pinpoint the pathways regulated by the gene products or chemical compounds being studied in your laboratory The Cignal Finder Arrays consist of 10 dual luciferase reporter assays and are designed for use in one of four research areas The targeted research areas are cancer immunology development and toxicology In this era of post genomics life science research many labs are investigating how diverse signal transduction pathways function on their own and in combination within the cell The Cignal Finder Arrays equip life science researchers to carry out such studies with speed and confidence The arrays are delivered in 12 tube strips including important negative and positive controls The assays are used right out of the box for the transfection or reverse transfection of the reporter assays into your cell lines of interest The
33. ive control expression vector 1 ul 100 ng Cignal positive control Mix each transfection cocktail gently 2 Prepare a SureFECT dilution for 9 tubes mentioned in step 1 by dispensing 2 7 ul of SureFECT into 225 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 25 ul of diluted SureFECT into each of the nine tubes containing 25 ul of diluted constructs 1 1 ratio as detailed in Table 4 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in a culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 CO2 Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately measure the cell density with a hemacytometer or automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specific complexes into the appropriate wells 7 Add 100 ul o
34. king the plate back and forth 8 Incubate cells at 37 C in a 5 COz incubator for 16 hours 9 After 16 hours of transfection change medium to assay medium Opti MEM containing 0 5 of fetal bovine serum 1 NEAA 100 U ml Penicillin and 100 ug ml Streptomycin Technical Support support SABiosciences com www SABiosciences com 24 Version 1 5 10 After 24 hours of transfection treat the cells as described in Table 12 with 1X 10X and 100X amount interfering peptide recombinant protein growth factor 1X is an smallest appropriate amount of small molecule or organic compound expected to modulate signaling pathway 11 To study the effect of interfering peptide recombinant protein growth factor we recommend harvesting cells 6 hours or 18 hours after treatment to develop luciferase assay 12 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay We found some components in DMEM interfere with SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that had been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM
35. ment p53 Hypoxia HIF response element Hypoxia inducible factor 1 HIF 1 NF B NF B binding element NF B Glucocorticoid Receptor Glucocorticoid response element GRE Glucocorticoid Receptor GR Cell Cycle pRb E2F E2F binding element E2F DP1 MAPK ERK Serum response element SRE Elk 1 SRF MAPK JNK AP 1 binding element AP 1 PKC Ca NFAT response element NFAT TGFB SMAD response element SMAD2 SMAD3 SMAD4 Myc Max E box binding element Myc Max Technical Support support SABiosciences com 28 www SABiosciences com Version 1 5 Notes Technical Support 888 503 3187 US 301 682 9200 29 Cignal Finder Reporter Arrays tube format Notes Technical Support support SABiosciences com www SABiosciences com 30 Version 1 5 Notes Technical Support 888 503 3187 US 301 682 9200 31 Cignal Finder 10 Pathway Reporter Arrays tube format Part 1034A Version 1 5 8 18 2008 fy SABiosciences Focus on Your Pathway BIOMOL GmbH Waidmannstr 35 A m 22769 Hamburg b m info biomol de 10 o www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D 888 503 3187 301 682 9200 www SABiosciences com support SABiosciences com
36. ntrol luciferase construct and constitutively expressing Renilla luciferase construct 40 1 1 NOTE These constructs are transfection grade and are ready for transient transfection These constructs are specifically designed to inhibit transformation and are NOT MEANT for introduction and amplification in bacteria Each Cignal Finder 10 Pathway Reporter Array includes the following components e Tubes 1 to 10 contain 10 different Cignal Pathway Reporters 100ng ul 2 X 50 ul e Tube 11 contains Cignal Reporter Assay negative control 100ng ul 2 X 50 ul e Tube 12 contains Cignal Reporter Assay positive control 100ng ul 2 X 50 ul Each strip is numbered from 1 to 12 Please refer to the specific Cignal Finder Array product specification sheet for the identity of each individual reporter assay in that array The strip cap used for each 12 tube strip has a tab attached to the cap for tube 1 This assists you in maintaining the caps in the proper orientation Technical Support support SABiosciences com www SABiosciences com 6 Version 1 5 B Description of Individual Cignal Reporter Assays Each Cignal Reporter Assay Kit includes the following components 1 Reporter Each reporter is a mixture of an inducible transcription factor responsive construct and constitutively expressing Renilla luciferase construct 40 1 The inducible transcription factor responsive construct encodes the firefly luciferase reporter gene under the
37. orter Assay System Promega Cat No E1910 This system requires cell lysis and is well suited for the rapid quantitation of both luciferase reporters when using luminometers with reagent auto injectors o Dual Glo Luciferase Assay System Promega Cat No E2920 This system is used to assay for both luciferase reporters on intact cells in growth medium This system can be used with any luminometer including those without reagent auto injectors 96 well white opaque flat bottom microtiter plate Luminometer Technical Support 888 503 3187 US 301 682 9200 9 Cignal Finder Reporter Arrays tube format IV Protocol A Before you begin 1 Cell line selection The Cignal Reporter Assay may be used with various mammalian cell lines Cell lines show a great deal of variation in the levels of signaling proteins The transcriptional activator activities in the cell line used will determine the sensitivity of the assay A cell line should be selected based on the functionality of the signal transduction pathway under investigation as well as for the transfectability of the cell line see below Transfection reagent selection We recommend the use of SureFECT SABiosciences Cat No SA 01 as a transfection reagent The Cignal Reporter Assay however also performs equally well with other transfection reagents such as Lipofectamine 2000 Invitrogen Cat No 11668 027 or FUGENE 6 Roche Cat No 1815091 When using al
38. ternative transfection reagents please refer to the manufacturer s instructions on the use of those reagents Optimization of transfection conditions The sensitivity of the Cignal Reporter Assay depends on the transfection efficiency The transfection efficiency in turn primarily depends upon cell line used Therefore it is very important to optimize the transfection conditions for each cell type under study Variables to consider when optimizing the transfection conditions include cell density cell viability amount of DNA ratio of DNA to transfection reagent transfection complex formation time and transfection incubation time see the detailed protocols for our recommendations The positive control construct included with each Cignal Reporter Assay can be used for determining the optimal transfection conditions Optimization of assay condition The response rate in the Cignal Reporter Assay depends on the assay conditions conditions of the experimental treatment To obtain maximum response given by any stimulus perform dosing and time course studies The optimal amount of stimulus and the time of treatment must be obtained empirically for each experiment see different protocols for our recommendations Important recommendations for best results A Perform all transfections in triplicate to minimize variability among treatment groups B Include positive and negative controls in each experiment to obtain reliable results C
39. ther vendors f you are using transfection reagent other than SureFECT follow their manufacturer s protocol for transfection The Cignal Reporter Assays also work well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the components in proportion to the surface area see section IV H This is just a general quideline the optimal conditions amounts should be adjusted according to the cell type and study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 3 Guidelines for setting up co transfections of a shRNA vector and Cignal Reporter Assay Table 3 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol Cignal Cignal Cignal Specific Negative Opti MEM SureFECT Opti MEM Time of Reporter Negative Positive shRNA Control Nucleic per well SureFECT transfection per well Control Control per well shRNA Acid Diluent hours per well per well per well Diluent per well per well 100 ng 1 1 0 ul 200 ng 25 ul 0 3 ul 25 ul 100 ng 2 1 0 ul 200 ng 25 ul 0 3 ul 25 ul 100 ng 3 1 0 ul 200 ng 25 ul 0 3 ul 25 ul 48 hor 72h 100 ng 4 1 0 ul 200 ng 25 ul
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