November 19 – Test and Final PCR Reactions (RADSeq)

This week we ran two PCR reactions. One was a test to see if it would work and the other was the final one for our DNA samples we have been working with.

I. PCR I (performed with inexpensive non-high-fidelity Taq)

NEB One- Taq 2x Master Mix 8.0 microliters 88.0 microliters
Forward Primer (10mM) 1-X 0.40 microliters 4.40 microliters
Reverse Primer (10mM) 2-6 0.40 microliters 4.40 microliters
Pure H2O 6.2 microliters 68.2 microliters
Master Mix Total 15 microliters 165 microliters

PCR was run using PCR1 on BIORAD #1/2

  • 94 degrees Celsius (30 sec)
  • 60 degrees Celsius (30 sec)
  • 68 degrees Celsius (45 sec)

Then it was put on a 4-10 degree Celsius infinite hold. The products were run on a 1.5% agarose gel with a 100 bp ladder at 130V for 40 minutes

[Successful amplification results in a. smear of fragments typically ranging from 50-700 base pairs] Ours was successful and was documented by Prof. Paul

Since the test was successful, we moved on to the final PCR reaction

II. Final PCR

Next we ran the final PCR. We made a different master mix for this part.

Phusion DNA Polymerase 0.31 microliters 3.41 microliters
5X Phusion HF buffer 6.25 microliters 68.75 microliters à 69
Forward Primer (10mM) 1.56 microliters 17.16 microliters à 17.2
Reverse Primer (10mM) 1.56 microliters 17.16 microliters à 17.2
DNTPs (10mM) 0.63 microliters 6.93 microliters
DMSO 0.94 microliters 10.34 microliters à 10.3
Pure H2O 10.75 microliters 118.25 microliters à 118
Master Mix Total 22.0 microliters 242.04 microliters

NOTE: We were assigned the PCR 2_1 for our master mix (assigned by Paul). For some reason we ran out of master mix when we were putting it in the samples so I (mistakingly) suggested that we use the next table’s master mix. They gave it to us and we used it BUT they had a different reverse primer :/ [they had 2_7]. The wrong master mix was used in reaction 21 and 22.

We ran this the same way and under the same conditions as the test PCR run (written above)


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