Supplemental Protocol 1: Generation of DNA fragments by PCR assembly of pooled oligos (NEB #M0689)
Each lab will have their own preference for how to produce genes of interest that can be further processed by Authenticase® for error correction. If you desire to take a “DIY” approach and choose to design oligos on your own followed by PCR to amplify the gene of interest, the following protocol may be helpful. Steps 1 and 2 are suggestions to generate PCR fragments less than 1 kb from your oligonucleotide design (each ≤ 60-mer) (Figure 6).
- Convert gene of interest into oligonucleotides less than 60 nt.
- Encode gene of interest in DNA manipulation software and break up into oligos of 60 nt or less.
- Order oligos from your preferred vendor or synthesize them in-house.
- Adjust each oligo to 10 µM to facilitate downstream processes.
- Encode gene of interest in DNA manipulation software and break up into oligos of 60 nt or less.
- Prepare gene-specific oligo pool.
- Transfer 5 µl of each oligo (10 µM) to a low bind microcentrifuge tube to form a pool of oligos encoding the gene of interest. Add nuclease-free water to a final volume of 500 µl (100 fmol/µl).
REAGENT
REACTION
FINAL RXN CONC.
Oligo 1.1 (10 µM stock)
5 μl
Oligo 1.2 (10 µM stock)
5 μl
…
Oligo 1.x (10 µM stock)
5 µl
Nuclease-free water
to 500 μl
100 nM of each oligo
- Setup PCR reaction:
REAGENT
REACTION
FINAL RXN CONC.
Q5 Hot Start High-Fidelity 2X Master Mix
25 μl
1X
10 μM Forward Primer
2.5 μl
0.5 µM
10 μM Reverse Primer
2.5 μl
0.5 µM
Template DNA (pooled oligos from 2.1)
5 µl
500 fmol of each oligo
Nuclease-free water
to 50 μl
- Setup PCR reaction conditions:
CYCLE STEP
TEMP
TIME
CYCLES
Initial Denaturation
98°C
2 minutes
Denaturation
98°C
10 seconds
36
Annealing
60–64°C*
10 seconds
Extension (for 500–700 bp)
72°C
40 seconds
Final Extension
72°C
5 minutes
Hold
4–10°C
* Please visit tmcalculator.neb.com to determine correct annealing temperature.
- Transfer 5 µl of each oligo (10 µM) to a low bind microcentrifuge tube to form a pool of oligos encoding the gene of interest. Add nuclease-free water to a final volume of 500 µl (100 fmol/µl).