Difference between revisions of "Luckau Protocols:PrimerResuspension"

 Primer Resuspension

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

• $\tfrac{ \mbox{(number of nmol)}}{x \mbox{ } \mu \mbox{L Low TE}} = \mbox{20} \mu \mbox{M}$
$x \mbox{ } \mu \mbox{L Low TE} = \tfrac{ \mbox{(number of nmol)}}{\mbox{20} \mu \mbox{M}}$

$\mu \mbox{M} = \tfrac{\mbox{pmol}}{\mu \mbox{L}} \bullet \tfrac{\mbox{nmol}}{1000 \mbox{ pmol}}$

$6 \mbox{ mm} \bullet \tfrac{0.1 \mbox{ } \mu \mbox{g ladder}}{1 \mbox{ mm width}}\ \bullet \tfrac{1 \mbox{ } \mu \mbox{L}}{1 \mbox{ } \mu \mbox{g}}\ = 0.6 \mbox{ } \mu \mbox{L ladder per well}$

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