Orthogonal cloning of clpXP: Difference between revisions

Revision as of 08:34, 13 July 2005

Clone clpX, clpP, YFP, YFP+ssRAtag (8peptide-LAA) into yeast. Use PCR on plasmids containing these genes. Clone these genes into different yeast plasmid shuttle vectors. Transform plasmids into yeast.
Test for expression of clpX and clpP by Western blot.
Test for functional protein degradation by clpXP. Determine if clpX is degrading proteins independent of clpP.
Check if yeast is surviving the addition of clpXP. This may have come up after step 1.
Measure rate of protein degradation via fluorescent signal of YFP.
Troubleshoot in unlikely event that it doesn't work perfectly the first time.

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---Steps:--
+==Steps:==
 #Clone clpX, clpP, YFP, YFP+ssRAtag (8peptide-LAA) into yeast.  Use PCR on plasmids containing these genes.  Clone these genes into different yeast plasmid shuttle vectors.  Transform plasmids into yeast.
 #Test for expression of clpX and clpP by Western blot.
 #Test for functional protein degradation by clpXP.  Determine if clpX is degrading proteins independent of clpP.
+#Check if yeast is surviving the addition of clpXP.  This may have come up after step 1.
+#Measure rate of protein degradation via fluorescent signal of YFP.
+#Troubleshoot in unlikely event that it doesn't work perfectly the first time.
-#Check if yeast is surviving the addition of clpXP.
+==Techniques to use==
+*PCR
+*Restriction enzyme use
+*Cloning into plasmids
+*Transforming yeasts
+*Culturing yeasts
+*Western blot
+*Fluorescence measurements
-#Measure rate of protein degradation via fluorescent signal of YFP.
+==Possible issues==
-#Troubleshoot in unlikely event that it doesn't work perfectly the first time.