see [[Genotyping DetailsProgram]] for strain specific details
==Materials==
# Dream Taq Green master mix
# Specific gene Primers (0.4um Working stock)
# Tail digest DNA
# ddH2O
==Protocol==
Use the following Volumes per 50ul Reaction:First, make 250ul of 0.4um working stock primers from 100um Primary Stocks.#248 ul ddH20#1ul forward primer (100um)#1ul reverse primer (100um)
#10X GoTaq Buffer: 5uL ("Molecular Biology Stuff" box in freezer)
#Primer Mix: 5ul
#dNTPs: 0.5uL of 10 mM ("Molecular Biology Stuff" box in freezer)
#Sterile water: 29ul
#Polymerase Go-Taq: 0.125uL ("Molecular Biology Stuff" box in freezer)
#Template: 1 uL
Master Mix (Per 5mL -- Make 1mL Aliquots)#10X GoTaq BufferUse the following Volumes per 25ul Reaction: 625uL ("Molecular Biology Stuff" box in freezer) #Primer Mix: 625ul#dNTPs: 62.5uL of 10 mM ("Molecular Biology Stuff" box in freezer)#Sterile water: 3625ul #Polymerase Go-Taq: 15.625ul ("Molecular Biology Stuff" box in freezer)*Add Template Individually
Run PCR Program Per sample (approx 2 hours1X)#Dream Tag Master mix: 12.5uL ("Molecular Biology Stuff" box in freezer) Use Cycler 1 on 6th Floor*Login#0.4um Primer Mix: Sergey, Just press enter to Login5ul*Under Genotype folder, pick Ingles program for Ingles genotyping*Under Genotype folder, pick regpcr program for PLT genotyping#Sterile ddH2O: 7.5ul
Make sure to press enter 2x once to confirm Tubes and second time to start *Template: 1 uL Run "specfic" PCRProgram for gene of interest (approx 2 hours). *[[Genotyping Program]]*[[PCR Amplification of DNA]]
see [[Preparing an Agarose Gel]] for details on preparing a DNA gel