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BioBank cDNA Kit TM Instructions for the use of BioBank cDNA in real-time PCR TM BB BioBank control cDNA Contents Introduction 3 Kit Contents 4 Reagents and Equipment to Be Supplied by User 4 Primerdesign Satisfaction Guarantee 5 Quality Control 5 Bench-side Protocol 6 Amplification Protocol 8 Primerdesign Ltd BioBankTM cDNA Published Date: 29-Jan-2016 2 Handbook HB09.01.04 Introduction The BioBank a high quality source of cDNA validated for use in real-time PCR experiments. The cDNA is reverse transcribed from high quality DNAse treated RNA from a variety of sources using an optimised blend of Oligo-dT and random nonamer primers. BioBank cDNA is therefore essentially free of genomic DNA and PCR inhibitors and covers the widest possible range of RNA and mRNA transcripts in the specified tissue or cell line. BioBank cDNA is useful for expression profiling of newly identified genes, and also as a positive control for the real-time PCR step. TM TM TM Positive control Primers Positive control primers and probe are supplied with the kits which detect 18S ribosomal RNA. These primers include a Double dye probe for optimal probe based detection through the FAM channel. The positive control primer and probe mix can be used with standard Taqman cycling conditions and with SYBRgreen Mastermix for detection using SYBR®green chemistry. The positive control primers will give a cycle threshold value of approximately CT=12 depending on the detection chemistry and the particular hardware settings in use. Primerdesign Ltd BioBankTM cDNA Published Date: 29-Jan-2016 3 Handbook HB09.01.04 Kit Contents BioBank cDNA 250ng (PURPLE) Control primers and probe for 18S ribosomal RNA (BROWN) RNAse/DNAse free water TM Reagents and Equipment to Be Supplied by User Real-Time PCR instrument Mastermix or Mastermix components This kit is designed to work well with all commercially available Mastermixes. However, we recommend the use of Primerdesign 2x PrecisionPLUS or PrecisionFASTTM Mastermix. TM Pipettors and Tips Vortex and centrifuge Kit Storage The Primerdesign BioBank cDNA kit should be stored at -20ºC on arrival. Freeze/thawing cycles should be kept to a minimum once resuspended. Under these conditions reagents are stable for six months from date of resuspension. TM Primerdesign Ltd BioBankTM cDNA Published Date: 29-Jan-2016 4 Handbook HB09.01.04 Primerdesign Satisfaction Guarantee Primerdesign takes pride in the quality of all of our products. Should this product fail to perform satisfactorily when used according to the protocols in this manual, Primerdesign will replace the item free of charge. Quality Control As part of our ISO9001 and ISO13485 quality assurance systems, all Primerdesign products are monitored to ensure the highest levels of performance and reliability. Primerdesign Ltd BioBankTM cDNA Published Date: 29-Jan-2016 5 Handbook HB09.01.04 Bench-side Protocol 1. Pulse-spin each tube in a centrifuge before opening. This will ensure lyophilised primer and probe mix is in the base of the tube and is not spilt upon opening the tube. 2. Resuspend lyophilised primer and probe mix and cDNA in RNAse/DNAse free water provided according to the table below; To ensure complete resuspension, vortex each tube thoroughly, allow to stand for 5 minutes and vortex again before use. Component BioBank cDNA (PURPLE) 18S control primers and probe (BROWN) TM Volume 130 µl 50 µl Real-time PCR 1. Positive control with 18S Make up a well in duplicate containing the positive control primers and BioBank cDNA TM Component PrecisionPLUS or PrecisionFAST Mastermix TM TM 18S primers and probe (BROWN) BioBank cDNA (PURPLE) RNAse/DNAse free water (WHITE) Final volume TM 1 Reaction 10 µl 1 µl 5 µl 4 µl 20 µl 2. Target gene with Primerdesign custom designed real-time PCR assay (optional step) Make up a well in duplicate containing the positive control primers and BioBank cDNA TM Component PrecisionPLUS or PrecisionFAST Mastermix Target gene primer mix BioBank cDNA (PURPLE) RNAse/DNAse free water (WHITE) Final volume TM TM Primerdesign Ltd BioBankTM cDNA Published Date: 29-Jan-2016 TM 1 Reaction 10 µl 1 µl 5 µl 4 µl 20 µl 6 Handbook HB09.01.04 3. Target gene with experimental primers (optional step) Make up a well in duplicate containing the experimental primers and BioBank cDNA TM Component PrecisionPLUS TM or PrecisionFAST Mastermix TM Forward primer Reverse primer Probe (optional) BioBank cDNA (PURPLE) RNAse/DNAse free water (WHITE) Final volume TM Primerdesign Ltd BioBankTM cDNA Published Date: 29-Jan-2016 1 Reaction 10 µl 6pmol 6pmol 3pmol 5 µl X µl 20 µl 7 Handbook HB09.01.04 Amplification Protocol Amplification conditions using Taqman or SYBR cycling settings with PrecisionPLUSTM Mastermix ® Cycling x40* ® Step Enzyme activation Denaturation DATA COLLECTION** Time 2min 10s 60s Temp 95ºC 95ºC 60ºC * For low copy number targets, giving late detection, a further 10 cycles may be needed to generate the complete amplification plot. ** Fluorogenic data should be collect during this step through the FAM channel. Amplification conditions using Taqman or SYBR cycling settings with PrecisionFAST Mastermix ® Cycling x40* ® Step Enzyme activation Denaturation DATA COLLECTION** TM Time 2min 5s 20s Temp 95ºC 95ºC 60ºC * For low copy number targets, giving late detection, a further 10 cycles may be needed to generate the complete amplification plot. ** Fluorogenic data should be collect during this step through the FAM channel. Primerdesign Ltd BioBankTM cDNA Published Date: 29-Jan-2016 8 Handbook HB09.01.04