Global UltraRapid Lentiviral Titer Kit
Contents
1. a 15 B Technical SUpport ienai ranana aena ea aea acna 16 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual Introduction and Background A Purpose of this Manual This manual describes a real time PCR based protocol to measure the copy numbers of integrated lentiviral constructs directly from lysates of the cells infected with SBI s lentiviral packaged constructs or libraries The protocol is based on amplification of a small fragment from the lentivector specific WPRE Woodchuck hepatitis virus Post transcriptional Regulation Element that is integrated into the genome of transduced cells The manual does not include information on packaging lentivector constructs into pseudotyped viral particles or transducing your target cells of choice with the particles This information is available in the user manual Lentivector Expression Systems Guide to Packaging and Transduction of Target Cells which is available on SBI s website www systembio com Before using the reagents and material supplied with this product please read the entire user manual B Determining Percentage of Cells Infected with Lentiviral Constructs Pantropic VSV G pseudotyped viral particles containing the lentivector expression construct can be used to efficiently deliver and stably express effector and reporter sequences in a wide range of mammalian target cells but transduction efficiency can vary signifi2. You need to determine the number of cells in a well of the plate upon infection For HT1080 cells the number of cells is around 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual 75 000 per well if you plate 50 000 cells in each well of a 24 well plate 24 hours before infection Three days after infection remove the medium and carefully wash the cells in each well with 1 mL of PBS Remove as much as possible all of the PBS from the wells Add 100 uL of lysis buffer to each well At this point you can either store the plate at 80 C until ready to proceed or quick freeze the plate in dry ice and then thaw the plate at RT Detach the cells in each well by flushing with the lysis buffer and pipetting up and down the cell suspension a few times Transfer as much as possible of the lysed cells into a PCR tube Gently pipette up and down a few times to break down any visible cell clumps Heat the lysate at 95 C for 2 minutes on a PCR machine Centrifuge the heated lysate at 14 000 RPM for 2 minutes and either put the tubes on ice or store at 20 C until ready to be used Run the qPCR as described for the Standard Curve calculations IMPORTANT If the cells have been transduced with unpurified pseudoviral stock directly using viral supernatant from 293 cells we recommend that after removing the medium containing the transfection reagent you wash the transduced cells 3 times w
3. Cat LV961A 1 Limited Warranty SBI warrants that the Product meets the specifications described in the accompanying Product Analysis Certificate If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price SBI s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2011 System Biosciences SBI 888 266 5066 Toll Free 650 968 2200 outside US Page 19
4. Figure 3 Example UCR Dissociation Curve 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual 4 000 GF 1 h i ADA fj vy i amei hi h i Figure 5 Example WPRE Dissociation Curve Page 12 ver 4 051611 www systembio com Global UltraRapid Lentiviral Titer Kit Cat LV961A 1 y 1 1921x R 0 996 Figure 6 Example Standard Curve 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual IV Troubleshooting A No PCR product Amplified No amplification from both calibration standard and sample DNA Repeat PCR and make sure you have added all the components in the master mix No amplification from sample DNA only The cells are not properly lysed Make sure the cells are washed carefully with PBS and all residue amount of PBS is removed from the wells B Dissociation Curves of Negative Controls are the Same as Those of Samples The negative controls are contaminated with a plasmid or sample containing WPRE in the lab Make sure you apply all the cautions of PCR set up to avoid contaminations Especially do not touch the inner lid of tubes always use filtered tips and avoid generate bubbles during pipetting C MOI Using an MOI that is too high This can result in an interferon response in the cells causing the cells to look ailing Using an MOI that is too low T
5. curve Set intercept at 0 check the Page 6 ver 4 051611 www systembio com Global UltraRapid Lentiviral Titer Kit Cat LV961A 1 boxes for Display Equation on chart and Display R squared value on chart e Calculate MOI for each of your samples using the equation For example if the equation you obtain from your experiment is y 1 192x and 2 of one of your samples is 5 1 the MOI of the sample should be 6 08 i e 1 192 multiplied by 5 1 IMPORTANT Please be aware that MOls for each standard provided may vary from lot to lot Refer to the tube of each standard for MOls of the particular lot B Titering Samples IMPORTANT The Global UltraRapid Lentiviral Titer Kit is compatible with both human and mouse cells Plate 50k cells well in a 24 well plate Use 3 wells in duplicate i e 6 wells Add 2 ul of concentrated virus in each of 2 wells Add 0 2 ul dilute concentrate 1 10 then pipette 2 ul into each of 2 wells Add 0 02 ul diluted concentrate 1 100 the pipette 2 ul into each of the 2 wells Transduce cells as indicated in the TransDux protocol Lyse cells after 72 hours C Lyse the Cells Transduced with Lentiviral Constructs Transduce HT1080 human NIH 3T3 mouse or your target cells of interest in a 24 well plate with packaged lentiviral construct or library using SBI s user manual Lentivector Expression Systems Guide to Packaging and Transduction of Target Cells
6. SSBI System Biosciences Global UltraRapid Lentiviral Titer Kit Cat LV961A 1 for Titering in Human and Mouse cells User Manual Store kit at 20 C on receipt A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement ver 4 051611 contained in this user manual Global UltraRapid Lentiviral Titer Kit Cat LV961A 1 Contents I Introduction and Background a 2 Purpose of this Manual e 2 B Determining Percentage of Cells Infected with Lentiviral COnstilicts O sa masi ata oa a A Seer ee 2 C Product Description and List of Components 4 D Additional Required Materials 5 Il Handling the Reagents in the Titering kit 5 Hl Prerok ares A A anand aaa ae 5 A Calculate a Standard Curve 5 B Titering Samples a 7 C Lyse the Cells Transduced with Lentiviral Constructs 7 D Pilot Experiment on Target Cells What is the Best MOI 9 IV Troubleshooting area 14 No PCR product Amplified eneee 14 B Dissociation Curves of Negative Controls are the Same as Those of Samples sra 14 Cie MO leapt uapa en A O et da 14 Ve ADDOnOIKC Su iu t tu au k leet vitae Du a th huku 15 A Related Producis
7. Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual PureFection Reagent Cat LV750A 1 Pure Fection Transfection Reagent is a new versatile and powerful polymer based gene delivery tool that ensures effective delivery of DNA into mammalian cells with low toxicity B Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http Avwww systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 265 North Whisman Road Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com Page 16 ver 4 051611 www systembio com Global UltraRapid Lentiviral Titer Kit Cat LV961A 1 V Licensing and Warranty Statement Limited Use License Use of the Lentivector Rapid Titer Kit e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions The Product shal
8. YBRTaq Mix and 1 uL of 25X Primer Mix for either UCR1 or WPRE Prepare two PCR master mixtures one for UCR1 and the other for WPRE enough for all reactions by doubling the volume of each ingredient with 2 plus the number of reactions Combine the required volumes of PCR Grade Water 2X SYBRTaq Mix and the Primer Mix in order Mix contents by inverting the tubes a few times and spin the tubes briefly in a microcentrifuge Aliquot 23 uL of the PCR Master Mix into each test tube or well if you are using a 96 well plate Add 2 uL of each of the six control DNA calibration standards or the cell lysates from Step A into the test tubes wells from Step 3 Seal the tubes or plate and place them in the real time PCR system Commence thermal cycling using the following program 50 C for 2 min 95 C for 10 min 95 C for 15 sec 60 C for 1 min for 40 cycles Add Dissociation step When the program is complete check the dissociation curve to make sure there is no significant contamination for WPRE amplification in the negative controls Then export Ct to an Excel file and calculate the average Ct of UCR1 and WPRE for each standard and sample e Calculate 2 where ACt Average Ct of WPRE Average Ct of UCR1 of the same standard or sample e Use the Excel software to plot the MOls of the standards against the values of 2 e Use the add trendline option of the software to draw the trendline of the standard
9. cantly depending on the transduction conditions and nature of target cells Therefore it is a standard procedure to determine the titer of the pseudovirus containing supernatant in control HT1080 human or NIH 3T3 mouse cells before proceeding with transduction experiments in your target cells After transduction of the lentiviral constructs into your target cells of interest it is also necessary to confirm the transduction efficiency of your experiments If a lentivector expression construct contains a GFP or RFP reporter the percentage of infected cells can be easily determined as the percentage of GFP or RFP positive cells by fluorescence activated cell sorting FACS However the procedure requires a FACS machine and it cannot be used if the vector does not contain a GFP or RFP marker Additionally the Page 2 ver 4 051611 www systembio com Global UltraRapid Lentiviral Titer Kit Cat LV961A 1 percentage of GFP or RFP positive cells does not always correlate with the number of infection competent viral particles present in your viral preparations This is because multiple viral particles can infect one single cell especially when infection is conducted at high MOls The relative titer concentration of a viral preparation is generally expressed as infection units ml IFU ml of infection competent pseudoviral particles The Global UltraRapid Lentiviral Titer Kit is designed to measure the titers of pseudoviral particles packa
10. ged with GeneNet siRNA libraries or any SBI lentiviral constructs by amplifying a fragment of WPRE directly from the lysates of infected cells It can be used to determine the copy number in cells transduced with any lentivector that contains the WPRE element regardless of the type of selection markers WPRE in SBI s lentiviral expression vectors and libraries enhances stability and translation of the internal promoter driven lentiviral transcripts It is integrated together with the lentiviral expression construct e g shRNA or cDNA into the genomic DNA of transduced cells Therefore the copy number of WPRE corresponds to that of the lentiviral expression constructs integrated into cells The kit contains calibration standards to measure titer which can be used to calculate MOI The calibration standards that are produced from WPRE containing genomic DNA have been extensively calibrated with cells infected with a copGFP reporter construct at different MOls By calculating the amounts of WPRE and the internal UCR1 control amplified from your samples and the calibration standards you can accurately determine the titer of the virus 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual Some key terms used in the protocol MOI multiplicity of infection The ratio of infectious pseudoviral particles ifu to the number of cells being infected IFU cells MOI IFU ml infectious units pe
11. his can result in an insufficient amount of construct to be expressed Page 14 ver 4 051611 www systembio com Global UltraRapid Lentiviral Titer Kit Cat LV961A 1 V Appendix A Related Products Lentivector Packaging Kits For FlV based Vectors pPACKF1 Cat LV100A 1 For HIV based Vectors pPACKH1 Cat LV500A 1 Unique plasmid mixes that produce all the necessary viral proteins and the VSV G envelope glycoprotein from vesicular stomatitis virus required to make active pseudoviral particles Producer Cell Line 293TN SBI Cat LV900A 1 transiently transfected with the packaging plasmids and an HIV based lentiviral construct produce packaged viral particles containing the lentiviral construct of interest 293TN Human Kidney Producer Cell Line SBI Cat LV900A 1 For packaging of plasmid lentivector constructs Packaged Positive Transduction Controls FlV based pSIF1 H1 siLuc copGFP Cat 4 LV201B 1 HIV based pSIH1 copGFP Cat LV600A 1 Packaged Positive control lentivectors allow you to measure transduction efficiency in target cells based on percent of GFP positive cells The H1 siLuc lentivector expresses an siRNA targeting Luciferase Transduction Reagent TransDux 200x Cat LV850A 1 Transdux is an optimized mix of cationic polymers used for efficient transduction of cells Each tube of Transdux provides enough material to transduce 80 wells in a 24 well plate format 888 266 5066
12. ith fresh media and 1 time with PBS to remove lentiviral plasmid DNA impurities These may be present in your cells due to residual transfer vector DNA from the 293 cell packaging step The number of viral particles in your viral suspension IFU ml can then be calculated with the following equation MOI of the sample X The number of cells in the well upon infection X 1000 ul of viral suspension added to the well for infection Page 8 ver 4 051611 www systembio com Global UltraRapid Lentiviral Titer Kit Cat LV961A 1 D Pilot Experiment on Target Cells What is the Best MOI Plate target cells approximately 50K per well in 6 wells of a 24 well plate Transduce with high MOls of 0 1 2 5 10 and 20 The MOI with the highest reporter gene expression and healthiest cells is the appropriate amount to use 888 266 5066 Toll Free 650 968 2200 outside US Page 9 User Manual System Biosciences SBI dowd YIN ez zz ojdwes ojdwes A tuud YIN Eu oz ejdues 6z gz ejduwes ejdwes 8 L Eos ojdwes ojdwes sssi is kanaa Si MOlOq p ull no SB S j9M OY 0 B eSA 99 JO piepue1s SU 1944 JO IT Z ppe usuL wawyd JYdM 10 1 dmas 193 YOdb p deyeyn Figure 1 Example qPCR Setup www systembio com ver 4 051611 Page 10 Global UltraRapid Lentiviral Titer Kit Cat LV961A 1 Amplification Pct 1 000 Ber eso ma mo me o e Temperature Ch es Co fc es fd ee
13. l be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research WPRE Technology SBI has a license to sell the Product containing WPRE under the terms described below Any use of the WPRE outside of SBl s Product or the Products intended use requires a license as detailed below Before using the Product containing WPRE please read the following license agreement If you do not agree to be bound by its terms contact SBI within 10 days for authorization to return the unused Product containing WPRE and to receive a full credit 888 266 5066 Toll Free 650 968 2200 outside US Page 17 System Biosciences SBI User Manual The WPRE technology is covered by patents issued to The Salk Institute for Biological Studies SBI grants you a non exclusive license to use the enclosed Product containing WPRE in its entirety for its intended use The Product containing WPRE is being transferred to you in furtherance of and reliance on such license Any use of WPRE outside of SBI s Product or the Product s intended use requires a license from the Salk Institute for Biological S
14. ltraRapid Lentiviral Titer Kit Cat LV961A 1 D Additional Required Materials For PCR Amplification Real time PCR System Recommended Applied Biosystems 7300 Real time PCR System Cat 4351101 ll Handling the Reagents in the Titering kit The reagents included in the titering kit are extremely sensitive to changes in temperature Please note the following storage and handling conditions The SYBR Taq mixture should be aliquoted immediately upon receipt and stored at 20 C The SYBR is light sensitive so please make sure to keep it in the dark for optimal performance The titering kit can be stored at 20 C for up to 1 year however once thawed the standards and primers should be kept at 4 C and should be used within one month The Standards primers and SYBR Taq are QCed and are lot specific for each lot of titering kit Please do not substitute other brands of SYBR Tag or mix and match standards and primers from other SBI kits and products lll Protocol A Calculate a Standard Curve Standards should be run in duplicate or triplicate so that an average of the ACt can be calculated The standard curve should be run at the same time as the samples that are being titered Please review all of the steps in the protocol before proceeding 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual 1 For each reaction you will need 9 5 uL of PCR grade water 12 5 uL of 2X S
15. r ml The relative concentration of infection competent pseudoviral particles Transduction Efficiency The average copy number of expression constructs per genome of target cell in the infected population C Product Description and List of Components The UltraRapid Lentiviral Titer Kit provides sufficient 2X SYBRTaq mix for 100 25 ul PCR reactions enough for a maximum of 42 individual and singleplex titers It also contains a cell lysis buffer that allows you to apply the cell lysates directly in the PCR reactions without the need for isolation and concentration measurement of genomic DNA The set of WPRE PCR primers is universal for any of SBI s HIV or FlV based lentivectors The Global Kit also includes the universal UCR1 primers as an internal reference for MOI calculation for both human and mouse lysates The calibration standards are genomic DNA samples isolated from cells transduced with a broad range of MOI using a copGFP packaged control construct Kit Components 50 ul 25X Forward and Reverse WPRE Primer Mix 50 ul 25X Forward and Reverse UCR1 Primer Mix 20 ul 12 5X Calibration Standard 0 Negative Control 20 ul 12 5X Calibration Standard 1 20 ul 12 5X Calibration Standard 2 20 ul 12 5X Calibration Standard 3 20 ul 12 5X Calibration Standard 4 20 ul 12 5X Calibration Standard 5 1 5ml 2X SYBRTaq Mix 5 ml Cell Lysis Buffer Page 4 ver 4 051611 www systembio com Global U
16. tudies This license agreement is effective until terminated You may terminate it at any time by destroying all Products containing WPRE in your control It will also terminate automatically if you fail to comply with the terms and conditions of the license agreement You shall upon termination of the license agreement destroy all Products containing WPRE in you control and so notify SBI in writing This License shall be governed in its interpretation and enforcement by the laws of California Contact for WPRE Licensing The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla CA 92037 Attn Office for Technology Management Phone 858 435 4100 extension 1275 Fax 858 450 0509 SBI has pending patent applications on various features and components of the Product For information concerning licenses for commercial use contact SBI Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Page 18 ver 4 051611 www systembio com Global UltraRapid Lentiviral Titer Kit
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