Last week, we ran DD-RADSeq and Ligation on our Erythranthe guttata samples. This week, we began by running a test run of PCR to test for successful library construction.

  1. I began by labelling new PCR tubes to correspond to our samples #9-16 for this new PCR run.
  2. We created a Master Mix for the table, enough for 11 reactions, using the following recipe:
  • NEB One-Taq 2x Master Mix: 8uL/rxn (88 uL total)
  • Forward Primer (10mM): 0.4 uL/rxn (4.4 uL total)
  • Reverse Primer (10mM): 0.4 uL/rxn (4.4 uL total)
  • Pure H2O: 6.2uL/rxn (68.2 uL total)

3. We added 15 uL of the Master Mix and 1 uL of the library DNA template to each corresponding PCR tube.

4. We added these PCR tubes to the thermocycler BIORAD #1/2 –>DDRAD –>2_PCR1. This runs at 94 degrees Celsius for 2 min, then 20 cycles of (94 degrees C for 30 sec, 60 degrees C for 30 seconds, 68 degrees C for 45 sec). 4-10 degree C infinite hold.

Gel Run

  1. After PCR was finished, we ran the PCR products in a 1.5% agarose gel (0.75 g agarose in 50 mL 1XTAE) with a 100 bp ladder at 130V for 40 minutes.

Final PCR

  1. To add Illumina flow cell annealing sequences, multiplexing indices, and sequencing primer annealing regions to all fragments and to increase concentrations of sequencing libraries, we perform a PCR amplification with a Fusion Polymerase kit.
  2. We prepared a Master Mix 2 for the table, enough for 11 reactions, following this recipe:
  • Fusion DNA polymerase: 0.31 uL/rxn (3.4 uL total)
  • 5X Fusion HF Buffer: 6.25 uL/rxn (68.8 uL total)
  • 10uM Forward Primer (PCR1_X): 1.56 uL/rxn (17.2 uL total)
  • 10uM Reverse Primer (PCR2_5): 1.56 uL/rxn (17.2 uL total)
  • 10mM dNTPs: 0.63 uL/rxn (6.9 uL total)
  • DMSO: 0.94 uL/rxn (10.3 uL total)
  • Pure H2O: 10.8 uL/rxn (118.3 uL total)

4. For each reaction, we combined 3 uL of the library template DNA to a new, labeled PCR tube.

5. We then added 25 uL of the Master Mix 2 to each tube.

6. We vortexed, and then spun down in the centrifuge.

7. We ran PCR2 on thermocycler BIORAD #2. Specifically, this cycle ran as:

Cycle Step Cycles Temp Time
Initial Denaturation 1 98 degrees C 30 sec



20 98 degrees C

65 degrees C

72 degrees C

10 sec

30 sec

30 sec (b/c 1kb genome)

Final Extension 1 72 degrees C 5 min
Hold 1 4 degrees C infinite


The next steps would be to run this PCR product on a gel and then select for bp size.