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| ==Protocol== | | ==Protocol== |
| Use the following Volumes per 50ul Reaction: | | Use the following Volumes per 25ul Reaction: |
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| #10X GoTaq Buffer: 5uL ("Molecular Biology Stuff" box in freezer) | | Per sample 1X |
| | #Dream Tag Master mix: 12.5uL ("Molecular Biology Stuff" box in freezer) |
| #Primer Mix: 5ul | | #Primer Mix: 5ul |
| #dNTPs: 0.5uL of 10 mM ("Molecular Biology Stuff" box in freezer)
| | #Sterile ddH2O: 7.5ul |
| #Sterile water: 29ul | |
| #Polymerase Go-Taq: 0.125uL ("Molecular Biology Stuff" box in freezer)
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| #Template: 1 uL
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| Master Mix (Per 5mL -- Make 1mL Aliquots)
| | *Template: 1 uL |
| #10X GoTaq Buffer: 625uL ("Molecular Biology Stuff" box in freezer)
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| #Primer Mix: 625ul
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| #dNTPs: 62.5uL of 10 mM ("Molecular Biology Stuff" box in freezer)
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| #Sterile water: 3625ul
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| #Polymerase Go-Taq: 15.625ul ("Molecular Biology Stuff" box in freezer)
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| *Add Template Individually | |
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| Run PCR Program (approx 2 hours).
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| Use Cycler 1 on 6th Floor
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| *Login: Sergey, Just press enter to Login
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| *Under Genotype folder, pick Ingles program for Ingles genotyping
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| *Under Genotype folder, pick regpcr program for PLT genotyping
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| Make sure to press enter 2x once to confirm Tubes and second time to start PCR
| | Run "specfic" PCR Program for gene of interest (approx 2 hours). |
| | * specific to each gene |
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| see [[Preparing an Agarose Gel]] for details on preparing a DNA gel | | see [[Preparing an Agarose Gel]] for details on preparing a DNA gel |