Luckau Protocols:PrimerResuspension

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Current revision (17:46, 12 March 2013) (view source)
 
Line 34: Line 34:
* <math>\tfrac{ \mbox{(number of nmol)}}{x \mbox{ } \mu \mbox{L Low TE}} = \mbox{20} \mu \mbox{M}</math>
* <math>\tfrac{ \mbox{(number of nmol)}}{x \mbox{ } \mu \mbox{L Low TE}} = \mbox{20} \mu \mbox{M}</math>
 +
 +
: <math>x \mbox{ } \mu \mbox{L Low TE} = \tfrac{ \mbox{(number of nmol)}}{\mbox{20} \mu \mbox{M}}</math>
: <math>x \mbox{ } \mu \mbox{L Low TE} = \tfrac{ \mbox{(number of nmol)}}{\mbox{20} \mu \mbox{M}}</math>
-
: <math>\mu \mbox{M} = \tfrac{\mbox{pmol}}{\mu \mbox{L}} \bullet \tfrac{\mbox{nmol}}{1000 \mbox{ pmol}}</math>
+
:::: note that: <math>\mu \mbox{M} = \tfrac{\mbox{pmol}}{\mu \mbox{L}} \bullet \tfrac{\mbox{nmol}}{1000 \mbox{ pmol}}</math>
 +
 
 +
 
 +
: <math>x \mbox{ } \mu \mbox{L Low TE} = \tfrac{ \mbox{(number of nmol)} \mbox{ (}1000 \mbox{ } \mu \mbox{L)}}{\mbox{20 nmol}}</math>
 +
 
 +
 
 +
: <math>x \mbox{ } \mu \mbox{L Low TE} = \mbox{(number of nmol)} \bullet 200</math>
-
<math>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===
+
===Resuspend!===
-
* 125µL dATP + 125µL dTTP + 125µL dCTP + 125µL dGTP + 4500µL H<sub>2</sub>O = 5000µL dNTP Mix @ 10mM (2.5mM each dNTP)
+
# spin down primer tube to ensure all primer crystals are at the bottom of the tube
-
* Aliquots: 1000µL into each of 5 snap-caps
+
# add Low TE, pH 8.0 (as calculated, see above)
-
:: 4 stored in freezer
+
# On the tube, write the concentration (20µM), then initial and date it
-
:: 1 stored in fridge for immediate use
+
# let tube sit overnight in fridge (4°C) to allow the crystals to fully dissolve into the buffer
 +
# store in fridge (4°C)

Current revision

Primer Resuspension

Tara K. Luckau's Home Page

Conservation Genetics Lab Notebook

Tara's Protocols


Contents

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}}


note that: \mu \mbox{M} = \tfrac{\mbox{pmol}}{\mu \mbox{L}} \bullet \tfrac{\mbox{nmol}}{1000 \mbox{ pmol}}


x \mbox{ } \mu \mbox{L Low TE} = \tfrac{ \mbox{(number of nmol)} \mbox{ (}1000 \mbox{ } \mu \mbox{L)}}{\mbox{20 nmol}}


x \mbox{ } \mu \mbox{L Low TE} = \mbox{(number of nmol)} \bullet 200



Resuspend!

  1. spin down primer tube to ensure all primer crystals are at the bottom of the tube
  2. add Low TE, pH 8.0 (as calculated, see above)
  3. On the tube, write the concentration (20µM), then initial and date it
  4. let tube sit overnight in fridge (4°C) to allow the crystals to fully dissolve into the buffer
  5. store in fridge (4°C)
Personal tools