In this lab, we tested for a successful library construction of Mimulus guttatus samples using a test PCR. The PCR was performed with inexpensive non-high-fidelity Taq. A master mix was created with 88 microliters of NEB One -Taq 2x, master mix, 4.4 microliters of Ilumina forward primer,4.4 microliters of the Ilumina reverse primer, and 68.2 of pure water. Once the master mix was created, separate reactions were done in PCR tubes for each Mimulus guttatus plant tissue. Along with 15 microliters of the master mix, we mixed in 1 microliter of the DNA restriction/ ligation product. We then ran PCR using PCR1 on BIORAD #1/2 thermocycler. We ran the products of PCR l for each sample on a 1.5% agarose gel (0.75 g agarose in 50 mL 1x TAE) with a 100 bp ladder at 130V for 40 minutes. We were able to see a smear of fragments, showing a successful library.
The second portion of this lab had a PCR run in which we added the special ‘special barcode’ sequences and the Ilumina primers to out libraries of Mimulus guttatus, allowing us to identify which specific individuals a given sequence comes from (Ligation barcode + PCR2 barcode). To generate the final Ilumina sequencing library, we began by adding flowcell annealing sequences, mutiplexing indices, and sequencing primer annealing regions to all fragments and to increase concentrations of sequencing libraries, we perform a PCR amplification with a kit. For each library, we set up 4-8 PCR reactions (to combine and mitigate PCR bias) in 50 microliter volume. Each PCR reaction contained ~20 nanograms (~3 microliters depending on concentration) of size-selected sample, PCR primers 1 and 2 at concentration 10 micromolar each, the recommended amount 5x-HF buffer, 10mM dNTPs, water, and DNA polymerase all in a standard 0.2mL PCR tube.