Lyophilised DNA mastermix handbook

Lyophilised DNA mastermix handbook
Techne qPCR lyophilised
2X qPCR MasterMix
Instructions for use of Techne Prime Pro lyophilised MasterMix
MM
MasterMix
Contents
Introduction
3
Kit Contents
4
Kit Storage
4
Suitable Sample Material
4
Licensing Agreement and Limitations of Use
4
Techne Satisfaction Guarantee
4
Quality Control
4
Bench-side Protocol
5
Reaction setup
6
Amplification Protocol
7
2
Techne Lyophilised MasterMix from Bibby Scientific
Introduction
Techne Prime Pro 2X lyophilised MasterMix is optimised for use in qPCR. The product is stable
at ambient temperatures for at least 18 months and can be conveniently shipped and stored at
room temperature. The MasterMix contains a thermo-stable TAQ Polymerase as well as buffer,
MgCl2 and stabilisers at concentrations optimised for the enzyme. The MasterMix has been
freeze-dried to produce a room temperature stable preparation.
The kit includes the lyophilised MasterMix, re-suspension buffer and a tube of ROX dye which
can be added as required when the MasterMix is to be used on hardware platforms that use ROX
as a passive reference dye.
The re-suspended solution requires only the addition of your cDNA, primers and probe to be
PCR ready.
The performance of Prime Pro lyophilised MasterMix is as good as or better than leading brands.
The perfect partner for Prime Pro kits
Techne Prime Pro 2X lyophilised MasterMix is designed for use with our range of Prime Pro
qPCR pathogen detection kits. Together they represent the ultimate solution for convenient
logistics as well as high quality qPCR performance.
3
Techne Lyophilised MasterMix from Bibby Scientific
Kit Contents
•
3 x Lyophilised MasterMix (50 reactions per glass ampule)
•
1 x Lyophilised ROX (BROWN)
•
4 x Re-suspension buffer (BLUE)
Kit Storage
The Prime Pro Lyophilised MasterMix should be stored at ambient temperature on arrival. The
kit is stable for at least 18 months at ambient temperature. Once resuspended in the provided
buffer the kit should be stored at -20°C. Repeated freeze/thawing will not compromise the
performance of the product. Under these conditions reagents are stable for six months from date
of resuspension.
Suitable Sample Material
All kinds of sample material suited for PCR amplification can be used. Please ensure the
samples are suitable in terms of purity, concentration and DNA integrity. Always run at least
one negative control with the samples. To prepare a negative-control, replace the test sample
with RNAse/DNAse free water.
Licensing Agreement and Limitations of Use
PCR is covered by several patents owned by Hoffman-Roche Inc. and Hoffman-LaRoche, Ltd.
Purchase of Techne kits does not include or provide licence with respect to any patents owned
by Hoffman-La Roche or others.
Techne Satisfaction Guarantee
Techne takes pride in the quality of all our products. Should this product fail to perform
satisfactorily when used according to the protocols in this manual, Techne will replace the item
free of charge.
Quality Control
As part of our routine quality assurance programme, all Techne products are monitored to ensure
the highest levels of performance and reliability.
4
Techne Lyophilised MasterMix from Bibby Scientific
Bench-side Protocol
1. For each glass ampule; Re-suspend lyophilised MasterMix in 525µl of re-suspension
buffer
Do not replace the re-suspension buffer with water or any other buffer.
The MasterMix is then ready to use as a 2X qPCR MasterMix
2. Add ROX if required
ROX is required for platforms that use ROX as a passive reference guide. Use table 1 below to
see if ROX addition is required for your hardware platform. If ROX is required then follow the
instructions below.
•
•
Re-suspend the Lyophilised ROX (BROWN) in the correct volume of re-suspension
buffer (BLUE) according to table 1 below.
Add re-suspended ROX to each ampule at the correct level.
Table 1. ROX addition
ROX resuspension
volume
ROX addition
per ampule
Applied Biosystems 7700, 7000, and 7900, 7300
StepOne, StepOnePLUS and ViiA7 platforms,
Roche capillary Lightcyclers.
100µl
20µl
All Stratagene platforms
200µl
15µl
Applied Biosystems 7500 platform
700µl
10µl
Real time PCR platform
All Other machines
NOT REQUIRED
NOT REQUIRED
5
Techne Lyophilised MasterMix from Bibby Scientific
Reaction setup
•
When using Techne Prime Pro pathogen detection kits.
For each 20µl real-time PCR reaction add the following to each reaction tube
Components
Prime Pro Lyophilised 2X qPCR
Primer/Probe mix
Template (25ng)
RNAse/DNAse free water
Final volume
•
1 Reaction
10 µl
1 µl
5 µl
4 µl
20 µl
Suggested use with user supplied primers and probe.
For each 20µl real-time PCR reaction add the following to each reaction tube
Components
1 Reaction
Prime Pro Lyophilised 2X qPCR
10 µl
Primers (6pmols Forward and Reverse)
x µl
Probe (3pmols)
x µl
Template (25ng)
x µl
RNAse/DNAse free water (up to Final volume)
x µl
Final volume
20 µl
*6pmols of primer gives a working concentration of 300nM in a 20µ l reaction
6
Techne Lyophilised MasterMix from Bibby Scientific
Amplification Protocol
•
For use with Prime Pro pathogen detection kits
Cycling x50
Step
Enzyme Activation - Hot Start
Denaturation
DATA COLLECTION*
Time
2 min
10s
60s
Temp
95ºC
95ºC
60ºC
*Fluorogenic data should be collected during this step according to the detection kit.
7
Techne Lyophilised MasterMix from Bibby Scientific
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement