Luckau Protocols:PCR

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PCR

Tara K. Luckau's Home Page

Conservation Genetics Lab Notebook

Tara's Protocols

Contents

Purpose

PCR, polymerase chain reaction, is used to amplify target fragments of DNA in vitro.

Reagents

  • template DNA (sample)
  • dNTPs
  • Taq polymerase (** MUST BE KEPT FROZEN! **)
  • Buffer (MgCl2, KCl)
  • primers

Consumables

  • PCR plate
  • pipet and tips

A note on pipets

  • (modified from Krystal's notes on µsat course):
  • One of the most critical technical aspects of this work is the proper manipulation of small volumes of liquid. Each pipet has an adjustable knob for setting the desired volume, a three-digit readout of the volume setting, and a tip-ejector knob at the back of the handle.
  • The table below highlights the volume range that each of these pipets span, and the details of interpreting the digital readout:
Pipet Range (µL) Min Volume Readout Max Volume Readout
P-2 0.1-2.0 0.10 2.00
P-20 2.0-20.0 02.0 20.0
P-200 20-200 020 200
P-1000 100-1000 010 100
  • Pipets should not be set or used beyond the range indicated above. Doing so will affect the calibration of the pipet, and, over time, will cause transfer of incorrect volumes.
  • To operate, dial in the desired volume, apply an appropriate tip, depress the plunger to the first stop and hold down, put tip in solution to be transferred, slowly release the plunger, watch the solution enter the tip, transfer to desired tube, and depress plunger again past the first stop (as a “blow-out” mechanism) to dispense. Eject tip into garbage.
  • The most critical steps here are letting the plunger release relatively slowly, and watching the solution enter the tip (some volumes are really tiny, but nonetheless visible upon inspection).

Protocol

Preparation

  1. fill ice bucket
  2. wipe work area down with 10% bleach

Master Mix

The PCR master mix is prepared to minimize low-volume pipetting as well as maintain consistency in reaction conditions between samples. Your master mix may contain any or all of the reagents listed above, depending on the PCR design. Refer to your PCR sheet. Typically, DNA template is pre-loaded into the PCR plate, then the master mix is prepared in a microtube then aliquoted among the wells containing DNA.

Important Notes

  • Taq must be kept frozen AT ALL TIMES!
  • All reagents except Taq should be flicked and briefly spun down
this ensures homogenization
  • Taq must NEVER be vortexed or flicked
this may denature the enzyme
  • Keep your PCR plate and master mix tubes on ice for the duration of set-up
the Taq we use for Team ConGen may begin working before we want it to if allowed to warm up. By keeping Taq and all related objects cold, we delay Taq activation

Thermal Cycler

  • Use PCR seal to cover your PCR reaction wells
  • Centrifuge plates (bottom of plate faces out)
allow centrifuge to get up to speed, about a minute
  • place securely in thermal cycler, top with silicone mat to prevent evaporation
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