The LightCycler System
1 BP-1.4-Sep-2003
Transfer Guidelines for LightCycler PCR Protocols from 20µl to 100µl
Reaction Capillaries
•The following pages contain general guidelines for transfer of established
20µl PCR protocols to 100µl. Recommendations of temperatures and times directly
refer to already existing protocols and may be different for de novo assay
development
•Some basic rules will be provided: they are intended to serve as starting point for
optimisation and establishment of 100 µl PCR protocols on LightCycler 2.0 instruments
•Further parameter-specific optimisation might be necessary to achieve
maximum performance for a distinct application
The LightCycler System
2 BP-1.4-Sep-2003
Transfer Guidelines for LightCycler PCR Protocols from 20µl to 100µl
Reaction Capillaries
1 Start with identical concentrations of enzyme, buffers, primers and probes
as used for 20µl PCRs
2 Check that capillary size is set to “100 µl“
3 Slopes of 20°C/s are recommended for each ramp during the thermocycling profile
- do not use other slopes to reach 95°C
- you might use any other slope for cooling to annealing temperature
and heating to elongation temperature
4 Set slope at 0.1°C/s or 0.05°C/s for melting curve analysis
The LightCycler System
3 BP-1.4-Sep-2003
Transfer Guidelines for LightCycler PCR Protocols from 20µl to 100µl
Reaction Capillaries
5 Set-up of a 100µl thermocycling profile
5.1 Initial Denaturation
•use a hold time of 60 seconds for regular Taq-DNA polymerase
•use a hold time of 10 minutes for FastStart Taq-DNA polymerase
5.2 Denaturation
• use hold times of 5-10 seconds for regular Taq-DNA polymerase
• use hold times of 15-20 seconds for FastStart Taq-DNA polymerase
5.3 Annealing
• add 25 seconds to the annealing time used for 20µl PCR
• lowering the annealing temperature by 1°C is recommended but
sequence/assay-dependent
5.4 Primer Extenison
• add 15 seconds to the elongation time used for 20µl PCR
6 Melting curve analysis
• apply identical settings as used for 20µl protocols
• be aware that Tm-values will be 0.3-1.1°C higher when the analysis is
carried out in 100µl capillaries
The LightCycler System
4 BP-1.4-Sep-2003
Transfer Guidelines for LightCycler PCR Protocols from 20µl to 100µl
Reaction Capillaries
20µl
100µl
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