PCR Analysis of Tail DNA: Difference between revisions

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-see [[Genotyping Details]] for strain specific details
+see [[Genotyping Program]] 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)
+Use the following Volumes per 25ul Reaction:
-#10X GoTaq Buffer: 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 (approx 2 hours).
+Per sample (1X)
-Use Cycler 1 on 6th Floor
+#Dream Tag Master mix: 12.5uL ("Molecular Biology Stuff" box in freezer)
-*Login: Sergey, Just press enter to Login
+#0.4um Primer Mix: 5ul
-*Under Genotype folder, pick Ingles program for Ingles genotyping
+#Sterile ddH2O: 7.5ul
-*Under Genotype folder, pick regpcr program for PLT genotyping
-Make sure to press enter 2x once to confirm Tubes and second time to start PCR
+*Template: 1 uL
+Run "specfic" PCR Program 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