Daniel:Notebook/ComboLock/2017-3-22
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Circular Product Production
The goal of this will be to produce a large amount of circular product for use in testing.
Protocol
- Ligation
- Make reaction mixture according to table; Do not add ligase yet
- Use following thermocycler program
- Heat mixture to 95C for 5 min
- Lower temp by 0.2C per second to 55C
- Hold mixture at 55C for 15 minutes
- Lower temp to 20C by reducing 0.2C per second
- Hold at 20C
- Add 1 uL T4 DNA ligase
- Incubate at 20C for 30 minutes
- TBE gel
- Mix uL TBE and uL 6X dye
- Aliquot 10 uL on to parafilm per sample/ladder
- Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to gel lanes
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
Reagent | Stock Conc | Final Conc./Amount | uL added |
T4 Ligase Buffer | 5X | 1X | 10 |
PosCon Circ Latch Oligo | 100 uM | 200 pmol | 1 |
Padlock 0601 oligo | 100 uM | 200 pmol | 1 |
C4 Positive Control oligo | 100 uM | 200 pmol | 1 |
C2 positive Control oligo | 100 uM | 200 pmol | 1 |
T4 DNA Ligase | 5 U/uL | 5 U | 1 |
nfH2O | NA | NA | 35 |
Total | 50 |