Home Protocols Supplemental Protocol 1: Generation of DNA fragments by PCR assembly of pooled oligos (NEB #M0689)

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).

  1. Convert gene of interest into oligonucleotides less than 60 nt.

    1. Encode gene of interest in DNA manipulation software and break up into oligos of 60 nt or less.

    2. Order oligos from your preferred vendor or synthesize them in-house.

    3. Adjust each oligo to 10 µM to facilitate downstream processes.

  2. Prepare gene-specific oligo pool.

    1. 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


    2. 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

      		  


    3. 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.