Download BioBank cDNA Manual

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.
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PrimerDesign Ltd BioBank
TM
cDNA
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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.
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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
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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
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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
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PrimerDesign Ltd BioBank
TM
cDNA
1 Reaction
10 µl
6pmol
6pmol
3pmol
5 µl
X µl
20 µl
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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.
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•
TM
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Amplification conditions using PerfectProbe cycling settings
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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