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
- Heat kill enzyme by incubating at 65C for 15 minutes
- Add 20 uL nfH2O
- TBE gel
- Mix 64 uL TBE and 16 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 Gel lanes
- 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 | 4 |
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 | 11 |
Total | 20 |
File:2017-03-22-CPProduction.png
Protocol Part 2
That was unexpected. From what I'm seeing on the internet, this might be a thing. Circular DNA looks to appear much higher on the gel than it should. I can try and Bglii digest to be sure.
- Bglii digest
- Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
- Make the following reactions; Do not add Bglii yet
- Use the following thermocycler program
- 5 min 95C
- Ramp to 50C at 0.2C/s
- 10 min 50C
- 1 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
- TBE Gel II
- TBE gel
- Mix 64 uL TBE and 16 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 Gel lanes
- 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 | Single Rxn Vol |
Ligation Reaction Product | 5 |
Bglii Cutter Primer (10 uM) | 5 |
10X Buffer 3.1 | 2 |
Bglii | 1 |
nfH2O | 7 |
Total | 20 |