Lentivirus qPCR Titer Kit - Applied Biological Materials
Contents
1. im Applied Biological Materials Inc 6 Tel 1 866 757 2414 Email info abmGood com Website www abmGood com Lentivirus GPCR Titer Kit Cat No Description Quantity LV900 Lentivirus QPCR Titer Kit 100 reactions Store at 20 C Assay Description and Application The Lentivirus qPCR Titer Kit provides a simple one step lentiviral vector titration This kit employs a quick RNA extraction step followed by qRT PCR assay which can be completed within 2 hours This kit is superior in both sensitivity and specificity and its non specific background is minimal compared to similar kits Kit Components Virus Lysis Buffer 800ul Reagent mix RT reagent amp Primer 1 0m STD1 lentivirus standard 1 300ul STD2 lentivirus standard 2 300ul Additional Materials Required not provided in the kit EvaGreen qPCR Mastermix appropriate for your particular instrument Please refer to our EvaGreen qPCR Mastermix Cat No Mastermix series for details Shipping and Storage Upon arrival Virus Lysis Buffer Reagent mix and STDs should be stored at 20 C Avoid repeated freeze thaw cycles to retain maximal performance Lentivirus GPCR Titer Kit is stable for 1 year from the ship date when stored and handled properly Protocol Overview Viral samples are collected from your virus producing cell line or purified viral prepara tions Emplyoing the virus lysis buffer provided in the kit viral RNA is isolated from a small aliquot of t2. StepOne system Temperature C Duration Number of Cycles 42 20min 95 10min 1 95 15sec eras n 40 60 Imin Data Analysis The titer of your samples can be calculated from Ct values by using our on line lentivi ral titer calculator at http www abmgood com viralexp lentiTiter dispOne ph Alternatively you could calculate the titer according to the following fomula Titer of unknown sample 5 x 107 28 Chcni ci2 ct Ctx Average of 3 Ct values of the unknown sample Ctl Average of 3 Ct values of STD1 Ct2 Average of 3 Ct values of STD2 ATTENTION Be sure to include the dilution steps if added in the calculation i e you diluted the virus sample 1 100 in Step 1 then the final titer will be 100 x 5 x 107 23 cr CH Ct2 Ct1 Recommendations for Optimal Result e Start the titration assay as soon as the virus is produced or thawed e Make aliquots of the reagent to avoid contamination e Start the PCR as soon as the reaction mixture is prepared and always keep the reaction mixture chilled in an ice box prior to PCR reactions For laboratory research only Not for clinical applications For technical questions please email us at technical abmgood com or visit our website at www abmGood com
3. he sample RT and primers included in the reagent mix are then used with the viral RNA the lentiviral STD1 and STD2 to determine threshold cycle Ct values via RT qPCR The titer of your samples can be determined by lenti titer calculation from resulting Ct values Protocol 1 Harvest lentiviral supernatant from cells and centrifuge it for 5 min at 2000g to remove cells and debris Please omit this step if you purchased the virus NOTE Please dilute your lentiviral stock 10 100 fold with IxPBS if the lentiviral sam ples underwent concentration and purification procedures Add 2ul of lentivirus sample to 18ul of virus lysis buffer Mix 10ul of the sample from Step 2 with 40ul of Reagent mix Mix 10uI STD1 with 40ul Reagent mix and 10ul STD2 with 40ul Reagent mix all in different tubes 4 Incubate the mixtures at room temperature for 3 min Prepare the reactions according to the tables below Please prepare reactions for each unknown sample and STDs in triplicate and one NTC reaction for nega ave controls Reagent mix without virus to ensure the system is not contaminated Samples STDs NIC D EvaGreen qPCR Mastermix 125W 125p 125W Lentivirus Sample Mixture from Step 3 125W i STD1 or STD2 Mixture from Step3 CSL o a Reagent mix ae Total per reaction 25W 25W 25W o Replication 3 ce x xT 6 Program the real time qPCR instrument for the following QRT PCR reaction cycles The conditions were optimized on ABI
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