MonthNovember 2019

DD-RADSeq PCR Test of Successful Library Construction

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.


We were able to digest out Mimulus guttatus samoles with selected restriction enzyme. We also utilized a digestion buffer appropriate for both enzymes. We placed 6 microliters of each sample’s DNA in the well of a PCR tube, and stored it on ice. We prepared a master mix with 9.9 microliters of CutSmart 10x buffer, 3.08 microliters of EcoRI-HF enzyme, 1.32 microliters of MPSI enzyme, and 18.7 microliters of pure water. To account for the multiple round of pipetting in the viscous liquid, we added 130% excess mastermix to each sample’s DNA. The total reaction volume amounted to 9 microliters and then the samples were sealed, vortexed, centrifuged and incubated at 37 degrees Celsius for 8 hours on a thermocycler with a heated lid set to 50 degrees Celsius.

In order to perform this double-digest restriction associated DNA sequencing, it it necessary to address adapter ligation. We first had to thaw the working stock EcoRI and MspI adapters that were made previously. We added 1 microliter of the working stock EcoRI adapter directly to each tube of digested DNA as follows: sample BT1 had Eco_2, sample BT2 had Eco_3, sample RM1 had Eco_4, sample RM2 had Eco_5, sample GS1 had Eco_6, sample GS2 had Eco_7, sample AH1 had Eco_8, sampled AH2 had Eco_9. A master mix was prepared with 4.8 microliters of CutSmart buffer 10x, 15.6 microliters of ATP, 2.4 microliters of T4 Ligase, 1.2 microliters of pure H2O, and 12 microliters of universal P2 MspI adapter (E). 130% excess of the mastermix was added to the tubes with the digested DNA and working stock to accommodate multiple round of pipetting with the viscous nature of the glycerol of the enzymes. We added 3 microliters of the mastermix to the digested DNA.

The total reaction volume of 13 microliters were sealed, vortexed, centrifuged, and incubuted at 16 degrees Celsius for 6 hours on a thermocycler with a heated lid set to 50 degrees Celsius. The samples were then stored frozen.

PCR Reaction with Plant Tissue DNA

In order to carry out the PCR reaction, we had to firstly prepare a PCR master mix for our group table, The Turtles. In the master mix, we mixed 200.4 microliters of distilled water, 30 microliters of 10x buffer, 30 microliters of MgCl2, 15 microliters of BSA, 3 microliters of dNTP’s, 3 microliters of each primer MgSTS332 primers- including both forward and backward- and .6 microliters of Taq into a sterile tube. This master mix was put in a sterile PCR tube with the extracted plant DNA that was obtained in a previous procedure. Each PCR tube that had plant DNA and master mix was labeled with group member’s initials and the initial label that appeared on the collection tube for each plant. Once all PCR tubes were filled and labeled, the Turtles group, along with the rest of the class was able to load the tubes into the PCR machine.

DNA Gel Electrophoresis with Plant Tissue

In this week’s lab, we obtained the DNA that was extracted from plant tissue previously and were able to run a gel electrophoresis analysis. The DNA was kept on ice until the procedure began. This included the dotting of purple dye on parafilm with a micropipette, using a singular tip, and added the some DNA to each dot with the same micropipette, but changing tips every time a new dot of DNA was added. Once the DNA was combined with the dye, we were able to use a micropipette that was set to a larger quantity to account for both the dye and DNA to transfer the colored DNA to the gel. We began by filling the first well with my samples and proceeded with each other member of the group, and then the last well was filled with the ladder by Professor Paul. The gel ran on a low voltage for an extended time in order to prevent running off on the gel.

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