Luckau Protocols:PrimerResuspension: Difference between revisions
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(New page: {|{{table}} width="900" |- | width="600" style="background-color: #BCED91;" align="center"| <span style="font-size:32px;"> Primer Resuspension </span> |align="left" style="background-colo...) |
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===Calculations=== | ===Calculations=== | ||
* Want: | * Want: 20µM primer | ||
* ( | * (number of nmol)/ | ||
:: (2.5mM) (5000µL) = (100mM) (V<sub>2</sub>) | :: (2.5mM) (5000µL) = (100mM) (V<sub>2</sub>) | ||
:: V<sub>2</sub> = 125µL of each dNTP | :: V<sub>2</sub> = 125µL of each dNTP | ||
Revision as of 12:58, 12 March 2013
| 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
- (number of nmol)/
- (2.5mM) (5000µL) = (100mM) (V2)
- V2 = 125µL of each dNTP
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
