Ensure you enter all PCR runs into the PCR form on the isolations database. You can use the form linked here to properly plan your PCR master mix and database entries. Use the database entry rows at the top to fill in your samples for one run, including a positive control (known working template for these conditions), an extraction negative control (negative control from extraction as template), and a PCR negative control (no template). Then put in the number of reactions in the master mix form, this will give you the necessary amount of reagents to make master mix for your reactions plus two additional.
It is recommended you input your PCR database entries before beginning mixing and your PCR run, this will reserve the spots so that the PCR IDs do not get taken while you are working.
Extract-N-Amp PCR ready premix
Typical master mix:
- PremixTAQ 12.5uL, found in freezer door. Fisher Catalog: RR003A
- Primer F (Conc. 10uM) 1uL
- Primer R (Conc. 10uM) 1uL
- PCR H2O, aliquots made by lab manager and stored in shared reagent box in door of small freezer. 9.5uL
- DMSO, aliquots made by lab manager and stored in shared reagent box in door of small freezer. 1uL
Please keep aliquots you open and use in your box after initial use.
Primer stock (100uM) locations can be found the Primer_locations table in the isolations database, these are in the ThermoFisher -80C freezer and will need to be diluted into a 10uM aliquot.
Multiply volumes by number of samples + 2 to make a small amount extra (form above does this). Use 25uL master mix and 1uL template per sample. PCR cycle will vary with primers used.
Common primers and cycle combinations used in our lab. Number in cycle title is usually annealing temperature.
- ITS1F/ITS4 – PCR_55_safe
- LR0R/LR3 – PCR_55_safe
- Bt2a/Bt2b – PCR_52
Another master mix:
- XNA template 4uL (if less, add XNA solutions up to 4 uL!)
- primer F 0.5 uL (ideally 0.4uM)
- primer R 0.5 uL (ideally 0.4uM)
- XNA PCR ready premix 10 uL
- water 5 uL (or add up to 20 uL total)
Typical cycling conditions:
- 1) 94C, 3 min
- 2) 94C, 45s
- 3) xC, 45s
- 4) 72C, 1:30min
- 5) go to 2 34x
NEB Q5 High Fidelity polymerase
High fidelity polymerases have 3′->5′ exonuclease activity, and can chew up primers. Solutions:
1) increase primer concentration
2) protect primers’ 3′ end by phosphorothioate (2 or 3 last bases, not just one).
Non-specific annealing solutions:
1) Optimize annealing temperature.
2) minimize the time that the enzyme has to amplify annealed primers by working on ice, preheating your thermocycler to96C, and using a Hot-Start option (probably not necessary for Q5 which is not active at room temp.)
Clonetech LA Taq and Buffer
|Clonetech Taq||0.25 ul|
|LA Buffer||2.5 ul|
|dNTPs (10mM each)||1 ul|
|primer F||1 ul|
|primer R||1 ul|
Start cycling with at least 94C for 1 min, can go up to 98C each cycle.