Amplification Of The Genetic Material; Required Materials - INGENASA INgene q IBV SYBR Instrucciones De Uso

8. AMPLIFICATION OF THE GENETIC MATERIAL

REQUIRED MATERIALS

Crushed ice.
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RNA extracted from the samples.
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Mixtures A, B and amplification buffer – KEEP IN CRUSHED ICE AT ALL TIMES.
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Positive amplification control A1 (IBV).
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RNAse-DNAse-free water.
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PROCEDURE
1. Prepare and identify as many tubes for the amplification as samples to be processed, adding an additional tube for
the positive amplification control, and another one for the negative control.
2. Take mixtures A, B and amplification buffer out from the freezer keeping them in crushed ice until thawing. Make
sure that they are correctly homogenised before taking out the required volume for the assay.
3. Prepare an appropriate amount of amplification mixture for the number of samples to be processed. The volume of
each reagent to be mixed for each of the samples is:
Per sample
For 10 samples
The tube used for mixing should be kept in crushed ice at all times. Likewise, it is recommendable to prepare an
excess amount of mixture (calculate an extra 10% for all reagents) in order to compensate for possible losses of
volume during pipetting.
4. Once mixture is prepared, homogenise correctly. Place the tubes previously labelled in crushed ice and add 20 µl of
the mixture prepared in this way to each tube.
Add 2 µl of previously extracted RNA samples to each tube, 2 µl of positive control A1 (amplification control for IBV),
to the corresponding tube and 2 µl of water to the tube labelled as negative control. Carefully mix the contents of
each tube and ensure that all the liquid has been deposited at the bottom of the tube. If not, tubes may be
centrifuged lightly until this occurs.
5. Following the thermocycler instructions, set the following conditions:
Retrotranscription
INSTRUCTIONS – ENGLISH
Mixture A
10 µl
100 µl
Temperature (ºC)
Amplification
Melting
Amplif. buffer Mixture B
10 µl 0.5 µl
100 µl 50 µl
Time
55 30 min
95 5 min
95 30 s
52 * 30 s
72 30 s
95 15 s
60 60 s
95* Continuous
Final Master Mix Volume
20.5 µl
205 µl
Cycles
1
40
1
(Versión 1410-14) – PAG.10.