Luckau Protocols:PCR

PCR	Tara K. Luckau's Home Page Conservation Genetics Lab Notebook Tara's Protocols NanoDrop PCR Agarose Gel Low TE Tris-Cl KCl

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

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

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