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BioBank TM cDNA Kit 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 Equipment to Be Supplied by User 4 PrimerDesign Satisfaction Guarantee 5 Quality Control 5 BenchBench-side Protocol 6 Amplification Protocol 8 PrimerDesign Ltd BioBank TM cDNA 2 Handbook 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 PerfectProbe for optimal probe based detection through the FAM channel. However the positive control primers and probe mix can also 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. TM PrimerDesign Ltd BioBank TM cDNA 3 Handbook Kit Contents • PURPLE) BioBank cDNA 250ng (PURPLE) • Control primers and probe for 18S ribosomal RNA (BROWN) BROWN) • RNAse/D NAse/DNAse free water TM Reagents and Equipment to Be Supplied by User • RealReal-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 Precision 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 BioBank TM cDNA 4 Handbook PrimerDesign 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 routine quality assurance programme, all PrimerDesign products are monitored to ensure the highest levels of performance and reliability. PrimerDesign Ltd BioBank TM cDNA 5 Handbook BenchBench-side Protocol 1. Pulse 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. Re Resuspend suspend lyophilised primer and probe mix and cDNA cDNA in RNAse/DNAse free water provided according to the table below; below; To ensure complete resuspension, vortex each tube thoroughly, allow to stand for 5 minutes, and vortex again before use. Component BioBank cDNA (PURPLE PURPLE) PURPLE 18S control primers and probe (BROWN BROWN) BROWN TM Volume 130 µl 50 µl RealReal-time PCR 1. Positive control with 18S Make up a well in duplicate containing the positive control primers and BioBank cDNA TM Component PrecisionTM Mastermix (not included) 18S primers and probe (BROWN BROWN) BROWN BioBank cDNA (PURPLE PURPLE) PURPLE RNAse/DNAse free water (WHITE WHITE) WHITE Final volume TM 1 Reaction 10 µl 1 µl 5 µl 4 µl 20 µl 2. Target gene with PrimerDesign custom designed realreal-time PCR assay (optional step) Make up a well in duplicate containing the positive control primers and BioBank cDNA TM Component PrecisionTM Mastermix (not included) Target gene primer mix BioBank cDNA (PURPLE PURPLE) PURPLE RNAse/DNAse free water (WHITE WHITE) WHITE Final volume TM PrimerDesign Ltd BioBank TM cDNA 1 Reaction 10 µl 1 µl 5 µl 4 µl 20 µl 6 Handbook 3. Target gene with experimental primers (optional step) Make up a well in duplicate containing the experimental primers and BioBank cDNA TM Component PrecisionTM Mastermix (not included) Forward primer Reverse primer Probe (optional) BioBank cDNA (PURPLE PURPLE) PURPLE RNAse/DNAse free water (WHITE WHITE) WHITE Final volume TM PrimerDesign Ltd BioBank TM cDNA 1 Reaction 10 µl 6pmol 6pmol 3pmol 5 µl X µl 20 µl 7 Handbook Amplification Protocol The 18S control primers are specifically formulated to work under all recommended realtime PCR cycling conditions. i.e. Taqman cycling, PerfectProbe cycling, and SYBR green cycling conditions will all work perfectly with the kit. ® • TM ® Amplification conditions using PerfectProbe cycling settings TM Step Enzyme activation Denaturation DATA COLLECTION* Extension Cycling x50 Time 10min 15s 30s 15s Temp 95ºC 95ºC 50ºC 72ºC *Fluorogenic data should be collected during this step through the FAM channel. • Amplification conditions using Taqman or SYBR green cycling settings ® ® Step Enzyme activation Denaturation DATA COLLECTION* Cycling x50 Time 10min 15s 60s Temp 95ºC 95ºC 60ºC *Fluorogenic data should be collected during this step through the FAM or SYBR channel. PrimerDesign Ltd BioBank TM cDNA 8 Handbook