Difference between revisions of "GoTaq PCR Genotyping"
From Bridges Lab Protocols
(Created page with '==Materials(100uM)== *GoTaq Master Mix *Primer Mix: Add 10uL of forward and reverse primer, 20uL total, into 1mL of dH20. *Reaction Mixture: 25uL GoTaq Mix, 10uL of Primer Mix, ...') |
Davebridges (Talk | contribs) (updated protocol and added categories) |
||
| (2 intermediate revisions by 2 users not shown) | |||
| Line 1: | Line 1: | ||
| − | ==Materials | + | ==Materials== |
*GoTaq Master Mix | *GoTaq Master Mix | ||
| − | *Primer Mix: Add 10uL of forward and reverse primer, 20uL total, into | + | *Primer Mix (from 100uM Primer Stocks): Add 10uL of forward and reverse primer, 20uL total, into 980uL of dH20 to make a 1uM solution. |
| − | *Reaction Mixture: 25uL GoTaq Mix, 10uL of Primer Mix, | + | *Reaction Mixture: 25uL GoTaq Mix, 10uL of Primer Mix, 15uL of template. If doing several samples make a master mix of primer mix + template (25 uL GOTaq + 10 uL Primer Mix)x # of Samples + 1 |
*Gel: 0.3g Agarose to 30mL of TAE, microwave until dissolved and add 1uL of EtBr. | *Gel: 0.3g Agarose to 30mL of TAE, microwave until dissolved and add 1uL of EtBr. | ||
| Line 12: | Line 12: | ||
*Add this mixture to each PCR tube. | *Add this mixture to each PCR tube. | ||
*Label and add each template to the corresponding PCR tube. | *Label and add each template to the corresponding PCR tube. | ||
| − | *Run PCR under | + | *Run PCR under appropriate [[Genotyping Program]] |
| − | *Load samples in gel and run on | + | *Load samples in gel and run on 130V (horizontally.) |
| + | |||
| + | [[Category:Mouse Work]] | ||
| + | [[Category:PCR]] | ||
| + | [[Category:Genotyping]] | ||
Latest revision as of 14:50, 6 December 2010
Materials
- GoTaq Master Mix
- Primer Mix (from 100uM Primer Stocks): Add 10uL of forward and reverse primer, 20uL total, into 980uL of dH20 to make a 1uM solution.
- Reaction Mixture: 25uL GoTaq Mix, 10uL of Primer Mix, 15uL of template. If doing several samples make a master mix of primer mix + template (25 uL GOTaq + 10 uL Primer Mix)x # of Samples + 1
- Gel: 0.3g Agarose to 30mL of TAE, microwave until dissolved and add 1uL of EtBr.
Procedure
- Prepare gel 30 minutes before PCR is finished.
- Prepare Reaction Mixture, adding 13x all materials except for the template.
- Add this mixture to each PCR tube.
- Label and add each template to the corresponding PCR tube.
- Run PCR under appropriate Genotyping Program
- Load samples in gel and run on 130V (horizontally.)
