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16S PCR

Theory

This protocol is for doing 16S rRNA polymerase chain reaction (PCR). This protocol has been adapted from the HUCK Genomics Core at Penn State University, University Park Campus.

Prepare Your Workspace

  • Turn on the PCR Clean Hood blower and white light.
  • Clean the cabinet with 70% ethanol including work surface and walls.
  • Turn the dial and run the UV light for at least 10 minutes. ⚠️ You cannot expect the glass to protect you from UV exposure. You can be in a different part of the lab while the light is running, but do not loiter in front of the cabinet.
  • Once the timer is up and the UV light turns off, you are ready to begin.

Remember, before placing any objects in the hood, they must be sprayed with 70% ethanol.

Gather Materials

  • Invitrogen Platinum SuperFi Master Mix (ThermoFisher Scientific, 12369050)
  • 515F Parrada Primer at 10uM (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTAA)
  • 806R Apprill Primer at 10uM (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACNVGGGTWTCTAAT)
  • Sterile PCR Water
  • PCR Plate/Tube Rack
  • Adhesive Film
  • 96-Well PCR Plate or 8-well PCR Tube Strip (depending on number of samples)
  • Micropipettes (P1000, P100, P10)
  • Pipette Tips (1000uL, 100uL, 10uL)
  • VWR Marker
  • Sterile 2.0mL Centrifuge Tubes

Master Mix Preparation

  1. In a sterile centrifuge tube labeled "Master Mix/MM" combine 10uL/sample Invitrogen Platinum SuperFi Master Mix, 0.4uL/sample 515F Parrada Primer, 0.4uL/sample 806R Apprill Primer, and 7.2uL sterile PCR water. You can use the 3.1a_PCR-Master-Mix-Workbook Excel file to calculate how much master mix to prepare based on the number of samples you have.

Table 1. Master mix preparation volumes per 20uL reaction

Reagent Volume
Invitrogen Platinum SuperFi Master Mix 10uL
515F Parrada Primer (10uM) 0.4uL
806R Apprill Primer (10uM) 0.4uL
Sterile PCR Water 7.2uL
  1. Vortex and spin down.

PCR Reaction

  1. Add 18uL of Master Mix to each PCR well.
  2. Add 2uL of extracted DNA to each respective well. Be sure to follow the plate map, create a new PCR plate map, and/or label tubes.
  3. Run thermocycler program as outlined in table 2.
  4. When cycles are complete, centrifuge tubes. Tubes can be stored at 4dC until ready to run gel. For longer term storage - store at -80dC.

Table 2. Thermocycler Parameters for Primary 16S PCR.

Cycle Time Temperature
Cycle 1 2 minutes 98°C
Cycle 2 10 seconds 98°C
Cycle 3 20 seconds 56.5°C
Cycle 4 15 seconds 72°C
Repeat Cycles 2-4 (30X)
Cycle 5 5 minutes 72°C
Hold at 4°C

Troubleshooting Your PCR