Setting up the reaction
Cool rotor and centrifuge bucket at -20 慢
Prepare stock reaction mix on ice. For each sample you will need
Water
Primers (stock with each 10 然)*
cDNA
2 x TaKaRa SYBR Premix Ex Taq**
* 1:1 mixture of 10 mM solutions of each of the two primers (stock concentration is therefore 5 mM of each primer)
** The protocol is for TaKaRa SYBR Premix Ex Taq. FastStartplus DNA Master SYBR Green I from Roche
is also a high quality mix, but more expensive.
Protect from light until needed.
Calculate one sample per cDNA to be analysed (initially you might want to convince yourself of the high precision of the LightCycler by running duplicates or triplicates), 8 samples for the standard curve (if you run consecutive runs you only need one standard in each of the following carousels), and two negative controls: one (cDNA-) negative control to which no cDNA is added and one (RNA-) negative control where no RNA was added to the reverse transcription reaction.
Insert rotor into centrifuge bucket and put the bucket on ice.
Insert the capillaries into the rotor
Pipet 8 無 of the reaction mix into each capillary.
Pipet 2 無 of cDNA into each capillary.
Cap the capillaries
Spin the rotor in the special centrifuge which comes with the LightCycler.
Start execution of the program using the following parameters:
Denaturation
Denaturation
Anneal/Ext.
Read absorbance
Perform melting analysis
Repeat 1-10 using the housekeeping primer pair.
