User Manual - Biomol GmbH
Contents
1. Our experimentally verified shRNA design algorithm assures gene specificity and efficacy An advanced specificity search in addition to BLAST built into the algorithm helps to reduce the potential off target effects At least two of the provided SureSilencing shRNA Plasmids is guaranteed to knock down expression of the targeted gene at the RNA level by at least 70 percent in transfected cells upon selection for antibiotic resistance or FACS based enrichment for GFP expression Benefits of the SureSilencing shRNA Plasmids e Enrich or Select Plasmids are available with either GFP Marker Hygromycin Neomycin or Puromycin Resistance enabling either enrichment and short term studies or selection and the study of the long term effects of gene suppression e GUARANTEED Knock down expression of any targeted human mouse or rat gene by at least 70 percent Control for non specific and off target effects e Convenient amp Cost Effective Use standard transfection methods Plasmids provide a renewable source of RNA Interference At least two of the four provided pre designed SureSilencing shRNA Plasmids are guaranteed to knock down expression of the targeted gene at the RNA level by at least 70 percent as measured by real time qRT PCR in transfected cells upon FACS based enrichment for GFP expression or selection for neomycin or puromycin resistance as described in this User Manual Technical Support support SABiosciences com www SAB2. flow cytometry core facility What transfection method should I use with the SureSilencing shRNA Plasmids We recommend using the SureFECT transfection reagent SABiosciences cat no SA 01 For the majority of cell lines tested it is an exceptional reagent providing a superior combination of maximal transfection efficiency with minimal cytotoxicity If you have previously optimized a transfection method for plasmids into your cell line that results in high transfection efficiencies you may use that method to transfect the SureSilencing shRNA Plasmids In order to optimize transfection conditions use a plasmid system that encodes an easily screened reporter such as GFP or beta galactosidase If your cells do not transfect well with lipid based or chemical transfection methods electroporation via methods such as those provided by Lonza Amaxa may be a useful alternative Technical Support 888 503 3187 US 301 682 9200 15 SureSilencing shRNA Plasmids Can I use the SureSilencing shRNA Plasmids with primary cells or macrophages or for injection into live animals No we do not recommend using the SureSilencing shRNA Plasmids with primary cells or macrophages or other cell lines that tend to be difficult to transfect with expression plasmids by traditional methods For these applications we instead recommend finding a viral based delivery system Similarly we do not recommend using the SureSilencing shRNA Plasmids for RNA interference in li
3. with the RT PCR Primer Sets d For convenient pipetting below dilute each completed 20 ul RT reaction 10 fold by adding 180 ul of ddH20 3 Primer Set and Master Mix Cocktail For real time PCR prepare two separate cocktails one for the GOI and one for the HKG using the following recipe Component Volume 2X SABiosciences Real Time PCR Master Mix 600 u ddH20 72 ul RT primer set for GOI OR HKG 48 ul Final Volume 720 ul 4 Set Up the Reactions Add 15 ul of the appropriate primer set and master mix cocktail and 10 ul of the appropriate diluted cDNA template RT reaction to the appropriate PCR wells as outlined in Figure 3 5 Perform Real Time PCR Perform PCR as described in RT Gene Expression Assays User Manual included with the RT PCR Primer Sets Technical Support support SABiosciences com www SABiosciences com 28 Version 1 8 6 Data Analysis An Excel based data analysis template that automatically performs the calculations below is available for download from our website at the following address http www SABiosciences com rnaidataanalysis php a Separately determine the average of the technical triplicate PCR C values and their standard deviations for both genes in each of the replicate transfections of each design and the negative control b Separately calculate individual AC values for each biological replicate transfection of each design and the negative control GOl
4. 10bp spacer 439 448 U1 termination sequence 449 465 SP6 RNA polymerase promoter 17 to 3 527 546 SP6 RNA polymerase promoter primer binding site 529 547 Binding region of pUC M13 reverse sequencing primer 564 585 SV40 early enhancer promoter 798 1216 SV40 minimum origin of replication 1114 1179 Puromycine N acetyltransferase coding region 1239 1838 Synthetic poly A signal 1883 1931 Beta lactamase Ampr coding region 2883 3743 Binding region of pUC M13 forward sequencing primer 4495 4518 T7 RNA polymerase promoter 17 to 3 4545 3 Insert Sequence The sense strand of the insert for each provided plasmid will read as follows 1 G only if the shRNA specific sequence does not already start with a G 2 The shRNA sequence listed on the individual product information sheet 3 CTTCCTGTCA the loop of the short hairpin RNA structure 4 The complementary sequence to the above shRNA sequence 5 CT to engineer a new diagnostic Pst site indicating the presence of insert This sequence is inserted between positions 438 and 439 in the plasmid sequence below directly in the middle of the bold underlined sequence of TCTC4GCAG The complete plasmid sequence can be accessed at http www SABiosciences com RNAiResource php Technical Support 888 503 3187 US 301 682 9200 23 SureSilencing shRNA Plasmids Appendix D Hygromycin Parent Vector Sequence pGeneClip Hygromycin Vector This vector can be obtained fr
5. 801 1550 1690 1822 1880 2563 2589 2637 3589 4449 5201 5224 5250 3 The sense strand of the insert for each provided plasmid will read as follows 1 G only if the shRNA specific sequence does not already start with a G oR WD This sequence is inserted between positions 438 and 439 in the plasmid sequence below The shRNA sequence listed on the individual product information sheet CTTCCTGTCA the loop of the short hairpin RNA structure The complementary sequence to the above shRNA sequence CT to engineer a new diagnostic Pst site indicating the presence of insert directly in the middle of the bold underlined sequence of TCTC GCAG The complete plasmid sequence can be accessed at http www SABiosciences com RNAiResource php Technical Support support SABiosciences com 22 www SABiosciences com Version 1 8 Appendix C Puromycin Parent Vector Sequence pGeneClip Puromycin Vector This vector can be obtained from Promega Corporation Madison WI Call one of the following numbers for ordering or technical information 1 800 356 9526 Outside U S 608 274 4330 linearized pGeneClip Puromycin Vector 4 56 bp Puromycin Synthetic 184 Polyadenylation Signal V40 Enhancer Early Promoter 4791MA pGeneClip TM Puromycin Vector sequence reference points Base pairs 4561 T7 RNA polymerase transcription initiation site 1 U1 promoter human 392 to 1 46 438
6. Specific snRNA AC AVG GOl Specific shRNA C GOI AVG GOl Specific shRNA C HKG GOl Specific shRNA AC STDEV STDEV 5 STDEV fke Negative Control shRNA AG AVG Negative Control shRNA C GOI AVG Negative Control shRNA C HKG Negative Control shRNA AC STDEV STDEVZ STDEV kko c Calculate the average AC and its standard deviation across the biological replicates for each design d Calculate the average AAC and its standard deviation for each design AA C Gene Specific shRNA AC Negative Control shRNA AC AA C STDEV STDEV2 STDEV ene Specific ACt NegativeControl ACt e Calculate the average knockdown and its 95 confidence interval Percent Knockdown 100 100 x 2 AAC Lower 95 Confidence Interval Boundary 100 100 x 2 AAC AA C STDEV Upper 95 Confidence Interval Boundary 100 100 x 2 AAC AA Ci STDEV f Interpretation Successful Design Observed KD gt 70 and an upper 95 C I boundary gt 55 5 Failed Design Observed KD lt 33 3 and a lower 95 C I boundary lt 55 5 Two out of the four designs should be successful Use at least two of the four pre designed plasmids that demonstrate the greatest percent knock down in your subsequent gene function assays and studies Technical Support 888 503 3187 US 301 682 9200 29 Notes Version 1 8 Notes Technical Support 888 503 3187 US 301 682 9200 31 SureSilencin
7. Txn2 Design1 Txn3 Design3 Txn2 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 D Design2 Txn1 Design3 Txn3 Design2 Txn1 Design3 Txn3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 E Design2 Txn2 Design4 Txn1 Design2 Txn2 Design4 Txn1 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 F Design2 Txn3 Design4 Txn2 Design2 Txn3 Design4 Txn2 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 G NC Design Txn1 Design4 Txn3 NC Design Txn1 Design4 Txn3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 H NC Design Txn2 BLANK NC Design Txn2 BLANK P1 P2 P3 P1 P2 P3 Samples in column 1 to 6 are amplified using Samples in column 7 to 12 are amplified PCR primers specific for the GOI using PCR primers specific for the HKG Technical Support 888 503 3187 US 301 682 9200 27 SureSilencing shRNA Plasmids Unless otherwise indicated follow the protocols described in the RT Gene Expression Assays User Manual included with the SABiosciences RT PCR Primer Sets 1 RNA Isolation a Isolate total RNA from each of the 15 transfections b For cultured cells use the Qiagen RNeasy Mini Kit Catalog 74104 Be sure to include the recommended DNase treatment step c Also make sure to perform the RNA quality control described in the RT Gene Expression Assays User Manual 2 Reverse Transcription First Strand cDNA Template Synthesis b Perform one reverse transfection for each of the 15 total RNA samples one per transfection c Follow the instructions in the RT Gene Expression Assays User Manual included
8. target gene but the target gene is required for the survival of the cells In other words the very act of suppressing the gene of interest itself killed the stably transfected cells To explore the function of essential genes you may need to develop and optimize an inducible promoter system for shRNA in you laboratory or seek other alternatives F Selection completed but low level of knockdown or stable transfection 1 Cells plated at too high a density during selection The use of too many cells during the selection reduces the effective concentration of neomycin Repeat the transfection and then repeat the selection at a lower cell density Preserve all of the transfected cells by dividing the entire population across a greater number of plates Conditioned medium may be used to promote growth at these lower densities Antibiotic concentration too low Be sure to use at least the minimum antibiotic concentration necessary to kill untransfected cells as defined by your kill curve Isolation of individual clones from pooled population is required to achieve maximal knockdown Choose the two pooled populations which exhibit the greatest level of knockdown Sub clone to select for single integration events Briefly dilute the cells such that a concentration of 0 5 cells per well of a 384 well dish is achieved Select 10 colonies from each of the pooled population to assay for knockdown Technical Support support SABiosciences com www S
9. 1 For 2RT Profiler PCR Arrays PA 011 12 For 12 RT Profiler PCR Arrays PA 011 24 For 24 RT Profiler PCR Arrays RT Real Time SYBR Green Specifically designed for instrumentation that does not require a reference dye BioRad MJ Research Opticon Opticon 2 and Chromo 4 Roche LightCycler 480 System Eppendorf Mastercycler ep realplex Instruments without ROX filter set Catalog Number Size PA 010 For 2RT Profiler PCR Arrays PA 010 12 For 12 RT Profiler PCR Arrays PA 010 24 For 24 RT Profiler PCR Arrays 3 RT PCR Primer Set targeting the suppressed target gene of interest and a housekeeping gene such as ACTB of GAPD to normalize the real time PCR results Technical Support support SABiosciences com www SABiosciences com 8 Version 1 8 IV Protocol A Transfection NOTES o Plasmid DNA purity and quality are crucial for optimal transfection efficiencies and therefore the success of shRNA based gene suppression o For guidance on carrying out transformations and plasmid preps and performing quality control testing on plasmid preps prior to transfection refer to Appendix E We recommend the use of SureFECT transfection reagent SABiosciences cat no SA 01 For virtually all cell lines tested SureFECT is an exceptional transfection reagent providing maximal transfection efficiency and minimal toxicity If you have already optimized a transfection reagent and protocol for your cell line of intere
10. A vector One 1 The negative control shRNA is a scrambled artificial sequence which does not match any human mouse or rat gene NOTE These plasmids are transformation grade ONLY and are prepared and meant for introduction and amplification in bacteria FIRST These plasmids ARE NOT transfection grade and ARE NOT provided in a large amount or of high enough quality for direct introduction into a mammalian cell line of interest Storage Conditions All components included with this catalog number are shipped with cold ice packs The plasmids must be stored at 20 C and are guaranteed for 6 months from the date received B Vector Information Plasmids contain either one of three markers identifiable by the catalog number scheme 1 Plasmids with catalog numbers ending in the letter H contain the hygromycin resistance gene for selection of stably transfected cells 2 Plasmids with catalog numbers ending in the letter N contain the neomycin resistance gene for selection of stably transfected cells 3 Plasmids with catalog numbers ending in the letter P contain the puromycin resistance gene for selection of stably transfected cells 4 Plasmids with catalog numbers ending in the letter G contain a gene encoding a Green Fluorescent Protein GFP for fluorescence microscopy based tracking or FACS based enrichment of transiently transfected cells The shRNA sequences and the negative control seq
11. ABiosciences com 18 Version 1 8 G Knockdown not distinguishable by real time PCR 1 Poor transfection efficiency and or no selection or enrichment Make sure that your transfection efficiency is optimized and that you have selected or enriched the cell population for transfected cells You must be looking at a population of cells that is nearly 100 percent transfected for an accurate determination of knockdown 2 Poor real time PCR reproducibility Make sure that your triplicate real time PCR determinations of threshold cycle values demonstrate a high degree of reproducibility with a standard deviation of roughly 0 25 to 0 33 cycles A seventy 70 percent knockdown of expression will be observed as only a 1 74 difference in normalized C values for the gene of interest between pure populations of negative control and gene specific shRNA transfected cells This specific level of reproducibility is required to reliably detect such a difference 3 Low level of expression of gene of interest To accurately determine knockdown by real time PCR the level of expression of the gene of interest in control or un transfected cells should be at least reasonably expressed with a C value less than 30 Real time PCR cannot determine the relative expression of genes expressed at a lower level C gt 30 with enough reproducibility to detect a seventy 70 percent knockdown of expression Try using more input RNA up to 5 ug in the reverse transcri
12. If using the GFP containing SureSilencing shRNA Plasmid Stain the cells with a nuclear DNA stain to count both transfected GFP positive cells and the total number of cells nuclear DNA stain in the same fluorescent view of the same microscope field Be sure to obtain numbers from several different randomly chosen microscope fields in the interior not toward the edges of the cell culture well NOTE DO NOT try to use the phase cell and fluorescence GFP views separately to estimate or count the total number of cells and the number of transfected cells respectively 4 If using a SureSilencing shRNA Plasmid set carrying an antibiotic resistance gene Use another plasmid instead with a reporter gene that allows you to count transfected cells Do not use one that relies on the total activity of reporter assay e g CAT in the entire cell population For example if using beta galactosidase fix and stain the cells with X gal to visualize and count the total number of cells and the number of transfected blue stained cells rather than assaying activity in total cell lysate D Interpreting knock down results from low transfection efficiencies 1 Select for antibiotic resistance or enrich by FACS for GFP expression to obtain a pure population of transfected cells For the GFP containing SureSilencing shRNA Plasmids ONLY The approximate apparent level of suppression in transfected cells is equal to the observed percent
13. N SABiosciences BIOMOL GmbH User Manual eat biomol Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D SureSilencing shRNA Plasmids Genome Wide Plasmid Based RNA Interference See Purchaser Notification for limited use license and warranty information pages 2 and 3 Part 1019A Version 1 8 07 01 2008 SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA SureSilencing shRNA Plasmids Genome Wide Plasmid Based RNA Interference User Manual For Catalog Numbers KX G H N P Weidmannstr 35 O biome info biomol de Ordering and Technical Service Contact Information Phoneras 40c8532600 or 0800 2466651 D 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 NOTICE TO PURCHASER I This product is made under license from The Carnegie Institution of Washington However
14. NOTES e Because plasmid DNA purity and quality are crucial for optimal transfection efficiencies and therefore the success of the shRNA based gene suppression we do not recommend using mini preparations of plasmid DNA for transfection e For guidance on carrying out transformations and plasmid preps and performing quality control testing on plasmid preps prior to transfection refer to Appendix E B For Transfection 1 Werecommend using SureFECT Transfection Reagent SABiosciences Cat No SA 01 2 Antibiotic Molecular Biology and or Tissue Culture Grade Hygromycin G418 for use with the Neomycin resistance plasmids or Puromycin Technical Support 888 503 3187 US 301 682 9200 7 SureSilencing shRNA Plasmids C For Real Time RT PCR Verification of Gene Suppression 1 RT PCR Array First Strand Kit Catalog Number C 02 2 SABiosciences RT Real Time SYBR Green PCR Master Mix Be sure to pick the correct one for the instrumentation in your laboratory RT Real Time SYBR Green ROX Specifically designed for All ABI and Stratagene Instrumentation Eppendorf Mastercycler ep realplex Instruments with ROX filter set Catalog Number Size PA 012 For 2RT Profiler PCR Arrays PA 012 12 For 12 RT Profiler PCR Arrays PA 012 24 For 24 RT Profiler PCR Arrays RT Real Time SYBR Green Fluorescein Specifically designed for BioRad iCylcer MyiQ and iQ5 Instrumentation Catalog Number Size PA 01
15. a frozen stock and for isolating total RNA Repeat the real time qRT PCR based validation of shRNA knockdown on selected clones from each plate Use one or more clones that demonstrate the greatest percent knock down from each of the two best designs in your subsequent gene function assays and studies Enrichment for GFP a Consult with your FACS instrument manufacturer or your local FACS core facility for details on enriching GFP expressing cells using this method Peak excitation of the GFP from the SureSilencing plasmids occurs at 505 nm with a shoulder at 480 nm and peak emission occurs at 515 nm b Please note that these peak excitation and peak emission wavelengths differ from those of other sources of GFP c Save the sort parameters as well as an image of the flow cytometer or FACS analysis trace for troubleshooting purposes d Be sure to enrich all cell populations those transfected with each gene specific shRNA plasmid and those transfected with the negative control shRNA Technical Support 888 503 3187 US 301 682 9200 13 SureSilencing shRNA Plasmids e Target gene suppression may also be analyzed at the individual cell level for example by immunofluorescence or morphology scoring The expression of the GFP from the SureSilencing plasmids can be monitored by fluorescence microscopy using an excitation filter of 470 20nm 470 40 nm and an emission filter of 515 nm long pass C Assay Effects of S
16. dd 3 ul of SureFECT that is 7 5 ul of SureFECT per ug of plasmid Mix by gently rocking the plate back and forth several times d Incubate the plate at room temperature for 20 minutes Technical Support 888 503 3187 US 301 682 9200 9 SureSilencing shRNA Plasmids e f zo During the above incubation prepare the cells 1 Wash the cells to be used with Dulbecco s PBS without calcium and magnesium trypsinize and harvest the cells by centrifugation 2 Wash once with cell culture media by resuspension and re centrifugation 3 Resuspend the cells in fresh growth medium containing 10 fetal bovine serum and 1 NEAA to a density of 1 6 X 10 cells per ml After the 20 minute incubation for transfection complex formation is completed step d above mix the tube containing the cells by inversion or gentle pipeting and aliquot 500 ul of the prepared cell suspension into the well containing the SureFECT plasmid complexes Mix gently by rocking the plate back and forth Incubate the cells at 37 C in a CO2 incubator for 24 to 48 hours Determine the transfection efficiency as the number of transfected cells divided by the total number of cells and begin the enrichment or selection process described in Section B Traditional Forward Transfection Protocol This is just a general guideline the optimal conditions amounts should be determined for each new cell line cell type being transfected a b One day before transf
17. e found in our white paper entitled Did Your RNAi Experiment Work Reliably Validating RNA Interference with Real Time PCR It may be downloaded from our website at the following address http www SABiosciences com manuals shRNAwhitepaper pdf For statistically significant results the method relies on triplicate transfections for each gene specific shRNA design and the negative control shRNA It also requires triplicate real time PCR reactions to characterize the targeted gene of interest GOI and a housekeeping gene HKG to normalize the results using the total RNA sample from each transfection Typical housekeeping genes include B actin and GAPDH Table 1 Setting up real time PCR validation of suppression The triplicate reactions for each gene in all five triplicate transfections may be conveniently setup in a 96 well PCR plate as depicted in this table The table represents a 96 well plate The reactions in the first set of six numbered columns will characterize expression of the GOI in the indicated RNA samples while the second set of six numbered columns will characterize expression of the HKG in the corresponding RNA samples 1 2 3 4 5 6 T 8 9 10 11 12 A Design1 Txn1 NC Design Txn3 Design1 Txn1 NC Design Txn3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 B Design1 Txn2 Design3 Txn1 Design1 Txn2 Design3 Txn1 P1 P2 P3 P1 R2 P3 P1 P2 P3 P1 P2 P3 C Design1 Txn3 Design3
18. ection seed 8 x 104 cells in each well of 24 well plate for roughly 30 to 35 percent confluence with 400 ul of growth medium On the day of transfection add 0 40 ug of each gene specific snRNA plasmid and the negative control shRNA plasmid into separate 50 ul aliquots of Opti MEM I Reduced Serum Medium Gibco Mix gently Prepare separate mixtures for each replicate well of cells to be transfected with the same plasmid For each well add 3 0 ul of SureFECT into 50 ul of Opti MEM that is 7 5 ul of SureFECT per ug of plasmid Mix gently and incubate all mixtures for 10 min at room temperature Add 50 ul of SureFECT mix to each 50 ul shRNA mix Mix gently and incubate for 20 min at room temperature Add each 100 pl mixture of shRNA and SureFECT in medium to the appropriate well containing cells and 400 ul of normal growth medium Mix gently Incubate the cells at 37 C in a CO2 incubator for 24 to 48 hours Determine the transfection efficiency as the number of transfected cells divided by the total number of cells and begin the selection or enrichment process described in Section B Our rigorous real time RT PCR protocol for verifying suppression by RNA interference relies on triplicate transfections for each gene specific shRNA design and the negative control shRNA for statistically significant results See Appendix F of this User Manual for more details Technical Support support SABiosciences com
19. g shRNA Plasmids SureSilencing shRNA Plasmids Part 1019A Version 1 8 07 02 2008 fy SABiosciences Focus on Your Pathway BIOMOL GmbH Waidmannstr 35 O 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 32
20. ilencing Gene Expression There are many ways to characterize the effects on cells brought on by a decrease in the expression of a gene meditated by RNA Interference The following is a brief list of possibilities Your experiments need not be limited to these suggestions however Cells may be harvested and RNA isolated for gene expression analysis using SABiosciences RT Profiler PCR Arrays SABiosciences RT Real Time PCR Primer Sets and SYBR Green Master Mixes As described in Appendix F verifying the suppression of gene expression SABiosciences GEArray Focused DNA Microarrays Cells may be harvested and protein isolated for SDS PAGE and Western Verification SABiosciences Multi Analyte Profiler ELISArray Kits SABiosciences Single Analyte ELISA Kits Biochemical Assays Cells may be left in wells or plates for SABiosciences Cellular Activation Signaling ELISA CASE Kits Cell biological assays such as morphology and immunofluorescence GFP only Steady state labeling or uptake assays neomycin or puromycin recommended Technical Support support SABiosciences com www SABiosciences com 14 Version 1 8 V SureSilencing FAQs What is the SureSilencing shRNA Plasmids guarantee We guarantee that at least two of the set of four SureSilencing shRNA Plasmids will knock down the expression of the target gene by at least 70 percent in transfected cells by real time qRT PCR in target shRNA transfected cells relati
21. included in the SureSilencing shRNA Plasmids Presently it is not clear under U S laws whether commercial users must obtain licenses from the owners of the rights to these U S patents before using SureSilencing shRNA Plasmids SABiosciences Corporation 6951 Executive Way Suite 100 Frederick MD 21703 USA CONTENTS l Background and Introduction 4 II Kit Contents and Vector Information 6 Ill Additional Materials Required T IV Protocol 9 A Transfection 9 B Selection or Enrichment 11 C Assay Effects of Silencing Gene Expression 14 V SureSilencing shRNA Plasmid FAQ 15 VI Troubleshooting Guide 17 Appendix A Neomycin Parent Vector Sequence Information 21 Appendix B GFP Parent Vector Sequence Information 22 Appendix C Puromycin Parent Vector Sequence Information 23 Appendix D Hygromycin Parent Vector Sequence Information 24 Appendix E Plasmid Preparation and Quality Control 25 Appendix F Technical Support support SABiosciences com 3 Real time RT PCR Protocol for Verifying Suppression 27 www SABiosciences com SureSilencing shRNA Plasmids I Background and Introduction RNA Interference a now commonplace and popular method for exploring gene function suppresses the expression of a specific gene of interest in transformed mammalian cell culture Upon suppression missing or altered activities in the cell can be attributed to the function of the affected gene However the most commonly used technique sma
22. iosciences com 4 Version 1 8 How It Works SureSilencing shRNA Plasmid Kit Contents 4 shRNAs targeting gene of interest 1 negative control shRNA ERB ZNG Ve ANNAN Plasmid Options For FACS Sorting For Stable Selection GFP Marker Cat Nos KX G Neomycin Cat Nos KX N Puromyin Cat Nos KX P Prepare stock of transfection grade plasmids by amplification in bacteria WW Transfect shRNA Plasmids into Cell Line of Interest Transfect all 4 shRNAs and Negative Control into your cell line in a six well culture plate 3 days shRNA1 shRNA 2 x 24 48 hours shPNA3 shRNA 4 Negative LA A A If using GFP Marker Plasmid Analyze Short Term Effects of Gene Suppression Enrich by FACS or analyze gene function by fluorescence microscopy gt 1day OR If using Selection Markers Analyze Longer Term Effects of Gene Suppression Select for stable transfectants using Neomycin or Puromycin 1 2 weeks Verify Suppression of Gene Expression Using Real Time PCR hours Perform biochemical cell or molecular biological assay of gene function Figure 1 Overview of SureSilencing shRNA Plasmid Procedure Technical Support 888 503 3187 US 301 682 9200 5 SureSilencing shRNA Plasmids Il Kit Contents and Vector Information A Kit Contents Component Specification Quantity shRNA SureSilencing shRNA vector Four 4 NC Negative Control shRN
23. knockdown divided by the experimentally determined transfection efficiency For example eighty 80 percent transfection efficiency and seventy 70 Technical Support 888 503 3187 US 301 682 9200 17 SureSilencing shRNA Plasmids percent observed knockdown 30 percent of control means an approximate percent suppression in transfected cells of 70 0 8 87 5 E No stably transfected cell population obtained after selection 1 Low transfection efficiency a Determine the observed transfection efficiency and optimize again if necessary b If you have not done so already repeat the plasmid purification using an endotoxin free plasmid purification kit particularly if excessive mammalian cell toxicity is observed upon transfection c Attempt to linearize the plasmid before transfection as described in this User Manual to help facilitate integration into your cell line s genome Antibiotic concentration too high Only use the minimum antibiotic concentration necessary to kill untransfected cells as defined by your kill curve Cells plated at too low a density during selection Plating cells at extremely low densities may inhibit growth due to the effective dilution of autocrine growth factors Conditioned medium may be used to promote growth or repeat the transfection and then the selection at a higher cell density Suppressed gene is an essential gene for viability The shRNA successfully suppressed the expression of the
24. ll interfering RNA siRNA proves useful for some applications but not all This technique works optimally with cells known to be easily and readily transfected with nucleic acid but not in cells with low transfection efficiencies Small interfering RNA does not allow transfected cells to be identified preventing both enrichment and determination of transfection efficiency Additionally due to a lack of selection markers siRNA only works under transient and not stable transfection conditions preventing the exploration of long term gene suppression effects The SureSilencing shRNA plasmids are designed using an experimentally validated algorithm These constructs specifically knock down the expression of specific genes by RNA interference and allow for enrichment or selection of transfected cells Each vector expresses a short hairpin RNA or shRNA under control of the U1 promoter and either the hygromycin neomycin or puromycin resistance gene or the GFP gene Hygromycin neomycin or puromycin resistance permits selection of stably transfected cells GFP helps estimate transfection efficiencies tracks transfected cells by fluorescence microscopy and permits FACS based enrichment of transiently transfected cells The ability to select or track and enrich shRNA expressing cells brings RNA interference to cell lines with lower transfection efficiencies Unlike siRNA plasmid based shRNA also provide a renewable source of RNA interference reagent
25. n the plasmid sequence below directly in the middle of the bold underlined sequence of TCTC GCAG The complete plasmid sequence can be accessed at http www SABiosciences com RNAiResource php Technical Support support SABiosciences com www SABiosciences com 24 Version 1 8 Appendix E Plasmid Preparation and Quality Control A coli Transformation 1 Use 2 ul of each stock plasmid solution to separately transform competent E coli cells following the manufacturer s protocol Store the remaining stock plasmid at 20 C NOTE n order to amplify a known amount of plasmid the E coli cells do not need to be extremely competent 2 Plate each transformation onto separate LB agar plates containing ampicillin Grow overnight at 37 C B Plasmid Purification 1 Pick one colony for each plate with a sterile loop or toothpick and inoculate separate 2 5 ml cultures of LB medium containing 50 ug ml ampicillin Incubate with shaking at 37 C until just a hint of turbidity is observed in the culture 3 to 6 h 2 Use the small cultures to inoculate 250 ml cultures of LB medium containing 50 ug ml ampicillin Incubate with shaking at 37 C overnight 3 Isolate the cells and purify plasmid DNA according to the Plasmid Purification Kit manufacturer s instructions for high copy number plasmids NOTE Save the remaining stocks of the plasmids provided by SABiosciences for future transformations and amplifications Howe
26. ninhibited growth should be observed from these plates d Replace the medium every 2 to 3 days with fresh medium Re plate cells every week Continue the selection for up to two weeks or until enough cells are available for generating a frozen stock and for isolating total RNA e As soon as possible be sure to freeze a stock of the stably transfected cells Be sure to generate stable transfections for each gene specific shRNA plasmid and the negative control shRNA plasmid f Once such a population is available continue to grow these cells in media containing a reduced or maintenance concentration of antibiotic typically 25 to 50 of the effective concentration 2 Special Note for Stable Transfection Applications Achieving a high level of knockdown in the initial population of stably transfected cells can be difficult The plasmid integrates into the cell line genome randomly and the initial polyclonal population represents several such integration sites Each integration site affects the relative level of expression of the shRNA construct differently and therefore the effectiveness of the knockdown Some sites provide more expression of shRNA and better knockdown of the GOI than others The level of knockdown in the polyclonal population represents the weighted average knockdown of all of the integration sites To obtain an even greater and more consistent level of knockdown this polyclonal population of stably
27. om Promega Corporation Madison WI Call one of the following numbers for ordering or technical information 1 800 356 9526 Outside U S 608 274 4330 Amp linearized pGeneClip Hygromycin Vector 4 989bp Hyaromycin Synthetic Polyadenylation SV40 E vancer Siona pD A Signa Early Promoter 4789MA pGeneClip TM Hygromycin Vector sequence reference points Base pairs 4989 T7 RNA polymerase transcription initiation site 1 U1 promoter human 392 to 1 46 438 10bp spacer 439 448 U1 termination sequence 449 465 SP6 RNA polymerase promoter 17 to 3 527 546 SP6 RNA polymerase promoter binding site 529 547 Binding region of pUC M13 reverse sequencing primer 564 585 SV40 early enhancer promoter 798 1216 SV40 minimum origin of replication 1114 1179 Hygromycin phosphotransferase coding region 1251 2276 Synthetic poly A signal 2311 2359 Beta lactamase Ampr coding region 3311 4171 Insert Sequence The sense strand of the insert for each provided plasmid will read as follows 1 G only if the shRNA specific sequence does not already start with a G The shRNA sequence listed on the individual product information sheet CTTCCTGTCA the loop of the short hairpin RNA structure The complementary sequence to the above shRNA sequence CT to engineer a new diagnostic Pst site indicating the presence of insert oR WP This sequence is inserted between positions 438 and 439 i
28. ption reaction and only dilute the completed reaction by four fold adding 60 instead of 100 ul of ddH20 but still use 10 ul of the dilution to setup PCR 4 Real time PCR analysis not performed properly Make sure that real time PCR analysis was set up and performed properly Consult the Troubleshooting Guide of the RT Gene Expression Assay User Manual if using the RT PCR Primer Sets and RT Real Time SYBR Green PCR Master Mixes from SABiosciences If using other reagents for real time PCR consult the original manufacturers recommendations and suggestions Note Only use real time PCR to determine the extent of knock down No other RNA detection method e g Northern analysis or conventional PCR will be quantitative enough to observe a 70 percent knockdown Western analysis is also unreliable because the success of knockdown at the protein level also depends on the quality of the antibody and the biological half life of the protein whereas RNA interference specifically acts at the RNA level See below Technical Support 888 503 3187 US 301 682 9200 19 SureSilencing shRNA Plasmids H Real time PCR decrease in expression at RNA level observed but no effect seen at the level of protein or biochemical assay The RT PCR verification will confirm that expression has been decreased at the level of messenger RNA and that the SureSilencing shRNA Plasmids functioned correctly A change in the RNA level for a particular gene product doe
29. s not necessarily immediately correlate with a change in the amount of protein in the cell If the protein has a long half life then changes in protein level will take much longer to occur than changes in the RNA level The protein level changes may therefore not be observed in a GFP based transient transfection experiment but should be detected in a longer term stable transfection based on hygromycin neomycin or puromycin selection Note Most mammalian expression plasmids like the SureSilencing shRNA Plasmids do not replicate in mammalian cells They are eventually lost due to dilution caused by cell growth and division causing random distribution to daughter cells The use of a selectable marker such as antibiotic resistance on the same or another plasmid at one tenth the amount selects for the very rare incorporation of the plasmid DNA into cell line genome by an unknown mechanism Once integrated the plasmid sequence is replicated with the rest of the genome and passed to both daughter cells Selection for the expression of GFP in such a fashion is not possible meaning that GFP may only be used for transient transfection experiments For any other troubleshooting or technical questions about the SureSilencing shRNA Plasmids please call one of our Technical Support representatives at 1 888 503 3187 or 301 682 9200 or email at Support SABiosciences net Technical Support support SABiosciences com www SABiosciences com 20 Ver
30. sion 1 8 Appendix A Neomycin Parent Vector Sequence pGeneClip Neomycin Vector This vector can be obtained from Promega Corporation Madison WI Call one of the following numbers for ordering or technical information 1 800 356 9526 Outside U S 608 274 4330 Amp sQOWOId LN linearized pGeneClip Neomycin Vector 4 758bp p g n Synthetic Poly en V40 Enhancer gna Early Promoter 4788MA pGeneClip Neomycin Vector sequence reference points Base pairs 4758 T7 RNA polymerase transcription initiation site 1 U1 promoter human 392 to 1 46 438 10bp spacer 439 448 U1 termination sequence 449 465 SP6 RNA polymerase promoter 17 to 3 527 546 SP6 RNA polymerase promoter primer binding site 529 547 Binding region of pUC M13 reverse sequencing primer 564 585 SV40 early enhancer promoter 798 1216 SV40 minimum origin of replication 1114 1179 Coding region of neomycin phosphotransferase 1251 2045 Synthetic poly A signal 2080 2128 Beta lactamase Ampr coding region 3080 3940 Binding region of pUC M13 forward sequencing primer 4692 4715 T7 RNA polymerase promoter 17 to 3 4742 3 Insert Sequence The sense strand of the insert for each provided plasmid will read as follows 1 G only if the shRNA specific sequence does not already start with a G The shRNA sequence listed on the individual product information sheet CTTCCTGTCA the loop of the short hairpin RNA s
31. st you may use that protocol to transfect the SureSilencing shRNA Plasmids into the same cell line Just be sure that the original protocol optimization used plasmids and determined transfected cells rather than relying on relative reporter activities We recommend using reverse transfection protocols with the SureFECT transfection reagent This is due to the time savings and improved reproducibility of using this method compared to traditional forward transfection methods SureFECT will also work well as a reagent for traditional forward transfection methods The following protocols are written on a per well basis and are designed for the transfection of an adherent cell line 293H Invitrogen with the SureFECT transfection reagent using a 24 well cell culture plate We recommend that you set up three 3 replicate transfections for each of the four gene specific and the negative control SureSilencing shRNA Plasmids using an optimized transfection protocol Reverse Transfection Protocol This is just a general guideline the optimal conditions amounts should be determined for each new cell line cell type being transfected a Dispense 100 ul of Opti MEM Reduced Serum Medium Gibco into the appropriate well of a 24 well cell culture plate b Dispense 0 40 yg of the appropriate gene specific shRNA plasmid or the negative control shRNA plasmid into the Opti MEM Mix by gently rocking the plate back and forth several times c A
32. th medium containing the following antibiotic concentrations in separate wells 0 100 200 400 600 800 and 1000 yg mI To generate a dose response curve for puromycin selection Plate untransfected cells at a low density lt 10 confluence in normal growth medium containing different puromycin concentrations in separate wells 0 1 2 4 6 8 and 10 ug mI a Allow the cells to grow until the O concentration point reaches confluence b Replace the media every 2 days during the selection process c Count the number of cells in each well and plot the cell number versus the antibiotic concentration d The minimum concentration of antibiotic that kills all of the cells is the effective concentration used for selection Technical Support 888 503 3187 US 301 682 9200 11 SureSilencing shRNA Plasmids To select transfected cells for antibiotic resistance in each plasmid transfection a After transfection re plate the cells at a low cell density lt 10 confluence b Grow cells in medium containing the effective concentration of antibiotic c Prepare control plates for all selection experiments to ensure that the selection conditions are still working as previously observed i Plate untransfected cells at the same density in medium containing the effective concentration of antibiotic No growth should be observed from these plates ii Plate transfected cells at the same density in medium without antibiotic U
33. the purchase of this material by a non academic or for profit organization will require a license to use the material from Carnegie License queries may be directed to Gloria Brienza or Gary Kowalczyk at The Carnegie Institution of Washington 1530 P Street NW Washington DC 20005 LIMITED PRODUCT WARRANTY This product is intended for research purposes only 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 II The purchase of SureSilencing shRNA Plasmids 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 shRNA to modify kit components for resale or to use SureSilencing shRNA Plasmids to manufacture commercial products without written approval of SABiosciences Corporation No other license expressed implied or by estoppel is granted U S patents may cover certain isolated DNA sequences
34. transfected cells can be cloned by limiting dilution to generate separate populations of stably transfected cells Each new clone or monoclonal population will represent a single integration site its level of shRNA expression and its level of GOI knockdown Screen for the clone or clones that provides the greatest level of knockdown using a real time qRT PCR procedure similar to the one described above in Appendix F Technical Support support SABiosciences com www SABiosciences com 12 Version 1 8 For example a Separately suspend a stock culture of the polyclonal populations of cells stably transfected with the two shRNA designs with the greatest apparent level of knockdown Determine their concentrations Choose one of the replicate transfections at random Serially dilute each suspended stock down to a concentration of roughly 1 cell per 400 ul Plate 200 ul of each dilution per well of separate 96 well cell culture plates so that roughly every other well will receive one cell Allow the cells to grow to large colonies in medium containing the maintenance concentration of the same antibiotic drug used for the first stage of selection hygromycin G418 or puromycin Re plate the each cell population separately into a larger well Allow them to grow to a larger number Re plate them again into an even larger well Continue this process iteratively until enough cells are available for generating
35. tructure The complementary sequence to the above shRNA sequence CT to engineer a new diagnostic Pst site indicating the presence of insert oR WN This sequence is inserted between positions 438 and 439 in the plasmid sequence below directly in the middle of the bold underlined sequence of TCTC GCAG The complete plasmid sequence can be accessed at http www SABiosciences com RNAiResource php Technical Support 888 503 3187 US 301 682 9200 21 SureSilencing shRNA Plasmids Appendix B GFP Parent Vector Sequence pGeneClip hMGFP Vector This vector can be obtained from Promega Corporation Madison WI Call one of the following numbers for ordering or technical information 1 800 356 9526 Outside U S 608 274 4330 r linearized pGeneclip hMGFP Vector 5 267bp Amp Pst 1638 pGeneClip hMGFP Vector sequence reference points Base pairs T7 RNA polymerase transcription initiation site U1 promoter human 392 to 1 10bp spacer U1 termination sequence SP6 RNA polymerase promoter 17 to 3 SP6 RNA polymerase promoter primer binding site Binding region of pUC M13 reverse sequencing primer CMV enhancer promoter Chimeric intron hMGFP open reading frame Synthetic poly A signal Beta lactamase Ampr coding region Binding region of pUC M13 forward sequencing primer T7 RNA polymerase promoter 17 to 3 Insert Sequence 5267 1 46 438 439 448 449 465 527 546 529 547 564 585
36. uence are provided with each product information sheet included with the plasmid set The sequences were cloned downstream of the U1 promoter of the plasmid For detailed information about the parent vectors including vector maps and sequences see Appendixes A B C and D Technical Support support SABiosciences com www SABiosciences com 6 Version 1 8 lll Additional Materials Required A For Transformation and Plasmid Purification 1 Competent E coli cells In general any strain designed for the amplification of plasmid may be used We recommend JM109 competent cells Promega Cat No L1001 or 2001 2 Ampicillin Molecular Biology grade from any source 3 LB medium and agar Gibco Recipe for LB ampicillin agar plates Add 15 g agar to 1 liter of LB medium Autoclave Allow the medium to cool to 50 C before adding ampicillin 50 ug ml final concentration Pour 30 35 ml of medium into 85 mm Petri dishes Let the agar solidify Store at 4 C for up to 1 month or at room temperature for up to 1 week 4 Plasmid Purification Kit such as a EndoFree Plasmid Maxi Kit Qiagen Cat No 12362 For purification of up to 500 ug transfection grade plasmid DNA OR b QIAfilter Plasmid Midi Kit Qiagen Cat No 12243 For fast purification of up to 100 ug transfection grade plasmid DNA PLUS EndoFree Plasmid Buffer Set Qiagen Cat No 19048 Endotoxin free buffer set for 10 mega or 5 giga transfection grade plasmid DNA preps
37. ut Figure 2 Example of Plasmid Preparation Quality Control Two SureSilencing shRNA Plasmids with GFP and their Pst digests are characterized side by side on an agarose gel along with a 1 kb ladder The Pst digests contain the two diagnostic bands indicating the presence of shRNA insert The uncut plasmid preparations predominantly contain the faster migrating supercoiled form and very small amounts of slower migrating nicked plasmid or plasmid concatamers 5 Optional Linearization of Plasmid for Stable Transfections To increase the likelihood of integration and shorten the time needed to isolate stable transfectants you may wish to linearize the plasmid containing the neomycin or puromycin resistance markers prior to transfection as described below a Perform a restriction enzyme digest on enough of your plasmid preparation for the necessary transfections with either Sca l Bsa or Dra Ill for the neomycin plasmids or with either Sca 1 EcIHK Nae or NgoM IV for the puromycin plasmids in order of preference b Re purify the DNA according to a standard phenol extraction procedure c Use the purified linearized plasmid DNA material to setup the stable transfection Technical Support support SABiosciences com www SABiosciences com 26 Version 1 8 Appendix F Real time RT PCR Protocol for Verifying Suppression A detailed description of the theory behind RNA interference validation using real time PCR may b
38. ve animals The SureSilencing shRNA Plasmids are meant for in vitro use only The delivery of any method of RNA interference into live animals to specific target tissues or organs is a difficult procedure for which no manufacturer has a viable solution Can the SureSilencing shRNA Plasmids be use for viral based delivery The SureSilencing shRNA plasmids cannot be directly used for viral delivery nor do they have convenient restriction sites to re clone the U1 based shRNA expression cassette into another vector or viral expression system In fact the U1 transcription termination sequence necessary for proper expression of the shRNA can disrupt the process of viral production Inserts for shRNA are more easily synthesized as oligonucleotides and then cloned into an appropriate expression system So instead we recommend screening the set of four shRNA sequences in another model system amenable to lipid mediated transfection to find the best sequence Then use the shRNA insert sequence information provided with the purchase of our plasmids to have the necessary oligonucleotides synthesized for cloning into your viral based delivery system of choice Do the SureSilencing shRNA Plasmids contain inducible promoters No SureSilencing shRNA Plasmids with inducible promoters are not available These promoters are meant to control the timing of silencing particularly for stable shRNA transfections for essential genes or for differentiation model systems Ho
39. ve to negative control shRNA transfected cells upon FACS based enrichment for GFP expression or selection for antibiotic resistance Please follow the recommendations in this User Manual in order to insure the optimal level of knockdown from these plasmids and the best method for detecting knockdown If you can demonstrate each plasmid s failure to knock down gene expression as described please contact a Technical Support representative to discuss your results and be prepared to provide the results as an Excel file in an email attachment If a product failure is verified we will send you another set of plasmids with four pre designed shRNA constructs for free Should I use the SureSilencing shRNA Plasmids with the GFP or antibiotic resistance marker Use the SureSilencing shRNA Plasmids with the antibiotic resistance marker for stable transfection to achieve long term knockdown a cell line permanently knocking down the gene of interest and to perform gene function assays involving the cell for a long period of time anything greater than a day Having multiple antibiotic resistance genes from which to choose enables you to introduce one or more SureSilencing shRNA into cells that are already resistant to an alternative antibiotic Use the SureSilencing shRNA Plasmids with GFP for transient transfections and gene function assays that can be performed with the cells quickly in less than a day but only if you have access and the resources to use a
40. ver use these remaining stocks only if your amplified plasmid preparation is compromised When the amplified stocks become depleted use your amplified preparation as the working stock solution both for transfections and for new transformations and amplifications Technical Support 888 503 3187 US 301 682 9200 25 SureSilencing shRNA Plasmids 4 Plasmid Quality Control and Diagnostic Restriction Digest To verify that you have purified plasmids containing shRNA insert you may perform a Pst restriction enzyme digestion of a small aliquot of each plasmid preparation All three plasmid types with either neomycin resistance or puromycin resistance or GFP will generate a diagnostic pair of bands on an agarose gel upon Pst digestion Parent Vector Pst Fragments SureSilencing Neomycin Vector 3827 bp and 991 bp SureSilencing Puromycin Vector 3209 bp and 1402 bp SureSilencing Hygromycin Vector 3892 bp and 1147 bp SureSilencing GFP Vector 4130 bp and 1197 bp To insure optimal especially transient transfection efficiency also characterize a small aliquot of each undigested plasmid on the same agarose gel The DNA should be predominantly supercoiled and not extensively nicked NOTE To avoid nicking the plasmid make sure the NaOH SDS lysis Solution I step of the plasmid preparation protocol does not proceed for any longer than the time specified O 1kbladder uncut Pstlcut uncut PstI c
41. wever they tend to be too leaky for RNA interference That is they still express a certain amount of the shRNA of interest even under repressed conditions We do not recommend a specific vector system for this application However you are welcome to screen our shRNA sequences in another cell model system and then use the shRNA insert sequence information provided with the purchase of our plasmids to have the necessary oligonucleotides synthesize for cloning into your inducible expression system of choice Technical Support support SABiosciences com www SABiosciences com 16 Version 1 8 VI Troubleshooting Guide A Poor plasmid transformation efficiency into E coli Consult the manufacturer s instructions for the component E coli cells B Poor yield of plasmid from transformed E coli Consult the manufacturer s instructions for the plasmid DNA purification kit C How to optimize or improve low transfection efficiencies 1 Transfection efficiencies primarily depend upon the 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 Do not use siRNA to optimize transfection efficiency
42. www SABiosciences com 10 Version 1 8 B Selection or Enrichment NOTE Unless your transfection efficiency is routinely greater than 90 we strongly recommend enriching the transfected cell population If you chose plasmids containing the hygromycin resistance gene select for transfected cells using hygromycin If you chose plasmids containing the neomycin resistance gene select for transfected cells using neomycin G418 If you chose plasmids containing the puromycin resistance gene select for transfected cells using puromycin If you chose GFP containing plasmids enrich the transfected cells by Fluorescence Activated Cell Sorting FACS 1 Selection for Antibiotic Resistance NOTE Different cell lines normally have different levels of antibiotic resistance Before transfection the minimum antibiotic concentration necessary to kill untransfected cells must be determined by generating a dose response curve The minimum concentration needed to kill untransfected cells is known as the effective concentration The effective concentration will depend on the cell line growth rate and state of confluence during growth In general more confluent cells tolerate higher concentrations of antibiotic therefore maintain the cells in a sub confluent state during selection To generate a dose response curve for either hygromycin or neomycin G418 selection Plate untransfected cells at a low density lt 10 confluence in normal grow
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