Cas12a (Cpf1) crDNA Template Synthesis
This is basically just PCR with a few minor changes. You should work under RNAse-free conditions since the product will be put directly into the T7 RNA transcription reaction overnight.
Contents
Section titled “Contents”1. Reaction setup
Section titled “1. Reaction setup”50 µL reaction volumes:
- Water: 33.5 µL H2O
- 5X Q5 Reaction Buffer: 10.0 µL
- 10 mM dNTPs: 1.0 µL
- 10 µM Forward Primer: 2.5 µL
- 10 µM Reverse Primer: 2.5 µL
- Q5 Polymerase: 0.5 µL
2. PCR program
Section titled “2. PCR program”- 98°C 0:05
- 10 cycles of:
- 98°C 0:05
- 57°C 0:30
- 72°C 1:00
- 72°C 2:00
- Hold at 4°C
3. Gel check and cleanup
Section titled “3. Gel check and cleanup”IMPORTANT: Have one separate control lane without polymerase. The differences in size between extended and non-extended DNA is small. Run a 3% TBE agarose gel with the control and all products, using NEB Low Molecular Weight ladder.
Do PCR cleanup with the MinElute Purification Kit. It’s like a regular PCR cleanup but optimized for short templates. Columns are in the door of the 4°C chemical fridge.