Download BioBank CDNA - Primerdesign Ltd

BioBank cDNA Kit
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Instructions for the use of BioBank cDNA in real-time PCR
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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.
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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
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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.
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
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.
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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)
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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
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Component
PrecisionPLUS or PrecisionFAST Mastermix
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18S primers and probe (BROWN)
BioBank cDNA (PURPLE)
RNAse/DNAse free water (WHITE)
Final volume
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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
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Component
PrecisionPLUS
or PrecisionFAST Mastermix
Target gene primer mix
BioBank cDNA (PURPLE)
RNAse/DNAse free water (WHITE)
Final volume
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Primerdesign Ltd BioBankTM cDNA
Published Date: 29-Jan-2016
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1 Reaction
10 µl
1 µl
5 µl
4 µl
20 µl
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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
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Component
PrecisionPLUS
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or PrecisionFAST Mastermix
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Forward primer
Reverse primer
Probe (optional)
BioBank cDNA (PURPLE)
RNAse/DNAse free water (WHITE)
Final volume
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Primerdesign Ltd BioBankTM cDNA
Published Date: 29-Jan-2016
1 Reaction
10 µl
6pmol
6pmol
3pmol
5 µl
X µl
20 µl
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Handbook HB09.01.04
Amplification Protocol

Amplification conditions using Taqman or SYBR cycling settings with PrecisionPLUSTM
Mastermix
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Cycling x40*
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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