BioBank TM cdna Kit. Instructions for the use of BioBank TM cdna in real-time PCR. BB BioBank control cdna

Transcription

1 BioBank TM cdna Kit Instructions for the use of BioBank TM cdna in real-time PCR BB BioBank control cdna

2 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 2

3 Introduction The BioBank TM 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 TM 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 TM cdna is useful for expression profiling of newly identified genes, and also as a positive control for the real-time PCR step. 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. 3

4 Kit Contents BioBank TM cdna 250ng (PURPLE) Control primers and probe for 18S ribosomal RNA (BROWN) RNAse/DNAse free water 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 TM TM or PrecisionFAS Mastermix. Pipettors and Tips Vortex and centrifuge Kit Storage The PrimerDesign BioBank TM 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. 4

5 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. 5

6 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 Volume BioBank TM cdna (PURPLE) 130 µl 18S control primers and probe (BROWN) 50 µl Real-time PCR 1. Positive control with 18S Make up a well in duplicate containing the positive control primers and BioBank TM cdna Component 1 Reaction PrecisionPLUS TM TM or PrecisionFAS Mastermix 10 µl 18S primers and probe (BROWN) 1 µl BioBank TM cdna (PURPLE) 5 µl RNAse/DNAse free water (WHITE) 4 µl Final volume 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 TM cdna Component 1 Reaction PrecisionPLUS TM TM or PrecisionFAS Mastermix 10 µl Target gene primer mix 1 µl BioBank TM cdna (PURPLE) 5 µl RNAse/DNAse free water (WHITE) 4 µl Final volume 20 µl 6

7 3. Target gene with experimental primers (optional step) Make up a well in duplicate containing the experimental primers and BioBank TM cdna Component 1 Reaction PrecisionPLUS TM TM or PrecisionFAS Mastermix 10 µl Forward primer 6pmol Reverse primer 6pmol Probe (optional) 3pmol BioBank TM cdna (PURPLE) 5 µl RNAse/DNAse free water (WHITE) X µl Final volume 20 µl 7

8 Amplification Protocol Amplification conditions using Taqman or SYBR cycling settings with PrecisionPLUS TM mastermix Cycling x40* Step Time Temp Enzyme activation 2min 95ºC Denaturation 15s 95ºC DATA COLLECTION** 60s 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 PrecisionFAS TM mastermix Cycling x40* Step Time Temp Enzyme activation 2min 95ºC Denaturation 5s 95ºC DATA COLLECTION** 20s 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. 8