Luckau Protocols:PrimerResuspension

Primer Resuspension

Tara K. Luckau's Home Page Conservation Genetics Lab Notebook Tara's Protocols

Purpose

Primers are used in polymerase chain reaction (PCR) to amplify a targeted region of genomic DNA. When ordered, primers arrive dry (in crystalized form), so, before use, the primer must be resuspended in liquid. In the Clark Lab, we resuspend primers to a concentration of 20µM (both unlabeled and labeled).

Materials

• dry primer (we currently use LifeTech: Invitrogen and AppliedBiosystems)
• each primer will be synthesized at a given amount of moles, usually in nanomoles (nmol) or picomoles (pmol) - find this information on the data sheets supplied by the primer manufacturer
• Low TE (pH 8.0)

Protocol

Calculations

• Want: 20µM primer

• [math]\displaystyle{ \tfrac{ \mbox{(number of nmol)}}{\mbox{x}}\ = \mbox{20} \mu \mbox{M} }[/math]

[math]\displaystyle{ \tfrac{ \mbox{(number of nmol)}}{\mbox{x}}\ = \mbox{20} \mu \mbox{M} }[/math]

(2.5mM) (5000µL) = (100mM) (V₂)

V₂ = 125µL of each dNTP

[math]\displaystyle{ 6 \mbox{ mm} \bullet \tfrac{0.1 \mbox{ } \mu \mbox{g ladder}}{1 \mbox{ mm width}}\ \bullet \tfrac{1 \mbox{ } \mu \mbox{L}}{1 \mbox{ } \mu \mbox{g}}\ = 0.6 \mbox{ } \mu \mbox{L ladder per well} }[/math]

Make dNTP Mix

• 125µL dATP + 125µL dTTP + 125µL dCTP + 125µL dGTP + 4500µL H₂O = 5000µL dNTP Mix @ 10mM (2.5mM each dNTP)
• Aliquots: 1000µL into each of 5 snap-caps

4 stored in freezer

1 stored in fridge for immediate use