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	=='''Protocols'''==		=='''Protocols'''==
-	===Mutations===	+	==First Week==
-		+
-	'''Ingredients'''	+
-		+
-	Site directed mutagenesis of 3 plant CYP450 (79A1, 79A2, 79B1).	+
-		+
-	We aim to destroy restrictionenzymes recognitionsites.	+
-		+
-	Primers was supplied by ...........	+
-		+
-		+
-	10 μl of 5× reaction buffer	+
-		+
-	X μl (50 ng) of dsDNA template	+
-		+
-	X μl (125 ng) of oligonucleotide primer #1	+
-		+
-	X μl (125 ng) of oligonucleotide primer #2	+
-		+
-	1 μl of dNTP mix	+
-		+
-	ddH2O to a final volume of 50 μl	+
-		+
-	Then add	+
-		+
-	1 μl of PfuTurbo DNA polymerase (2.5 U/μl)	+
-		+
-		+
-	'''Poly Chain Reaction'''	+
-		+
-	We ran a PCR to syntesise and amplify our mutated CYP's.	+
-		+
-		+
-	Cycling Parameters for the QuikChange Site-Directed Mutagenesis Method	+
-		+
-	Segment 1	+
-		+
-	Cycles 1 Temperature 95°C Time 30 seconds	+
-		+
-	Segment 2	+
-		+
-	Cycles 12	+
-		+
-	Temperature 95°C Time 30 seconds	+
-		+
-	Temperature 55°C Time 1 minute	+
-		+
-	Temperature 68°C Time 1 minute/kb of plasmid length	+
-		+
-		+
-	'''Digestion'''	+
-		+
-	We aim to remove the parentel CYP.	+
-		+
-	We take advantage of the fact that this CYP is methylated on cytosines. Dpn is a restriction enzyme that cuts DNA which is methylated - therefore our new mutated CYPs remain untouched.	+
-		+
-	1. Add 1 μl of the Dpn I restriction enzyme (10 U/μl) directly to each amplification reaction.	+
-		+
-	2. Gently and thoroughly mix each reaction mixture by pipetting the solution up and down	+
-	several times. Spin down the reaction mixtures in a microcentrifuge for 1 minute and	+
-	immediately incubate each reaction at 37°C (in a heater with lid or in a 37°C room) for 1 hour to digest the parental (i.e., the	+
-	nonmutated) supercoiled dsDNA.	+
-		+
-		+
-	'''Source'''	+
-		+
-	Adapted from	+
-	QuikChange™ Site-Directed	+
-	Mutagenesis Kit	+
-	INSTRUCTION MANUAL	+
-		+
-		+
-	===Competent Cells===	+
-		+
-	'''E. coli Calcium Chloride competent cell protocol'''	+
-		+
-	1. Inoculate a single colony into 5mL Lb in 15mL falcon tube. Grow	+
-	O/N at 37°C.	+
-		+
-	2. Use 1mL to inoculate 100mL of LB in 250mL bottle the next morning.	+
-		+
-	3. Shake at 37°C for 1.5-3hrs until OD600 = 0.4-0.8	+
-		+
-	Then….	+
-	1. Put the cells on ice for 10 mins (keep cold form now on).	+
-		+
-	2. Collect the cells by centrifugation in the big centrifugue for 10 mins	+
-	at 6krpm	+
-		+
-	3. Decant supernatant and gently resuspend on 10 mL cold 0.1M	+
-	CaCl (cells are susceptible to mechanical disruption, so treat them	+
-	nicely).	+
-		+
-	4. Incubate on ice x 20 mins	+
-		+
-	5. Centrifuge as in 2	+
-		+
-	6. Discard supernatant and gently resuspend on 5mL cold	+
-	0.1MCaCl/15%Glycerol (from a 85% stock)	+
-		+
-	7. Dispense in microtubes (300μL/tube). Freeze in -80°C.	+
-		+
-	'''Source:'''	+
-		+
-	Adapted from	+
-	http://www.med.nyu.edu/medicine/labs/blaserlab/Protocols/E-coli_competent_cells_protocol_&_transformation.pdf	+
-		+
-		+
-	===LBamp Plates===	+
-		+
-	1. 500 ml LB agar and 500 μL amphicilin	+
-	2. Pour on plates	+
-	3. Leave with the lid half on for 30 minutes at room temperature	+
-	4. Put in refrigerator until needed.	+
-		+
-	===Transformations===	+
-		+
-	'''Transformation of Ca++ competent cells'''	+
-		+
-	1. Put 10μL of circular plasmid or all of a ligation reaction of plasmid	+
-	DNA in a microtube. Gently add ~50μL of competent cells.	+
-		+
-	2. Incubate for 30 mins on ice.	+
-		+
-	3. Heat shock for 45 seconds at 42°C. Put back on ice.	+
-		+
-	4. Plate the whole lot in LBamp plates	+
-		+
-	5. Leave the plates at 37°C O/N	+
	==Second Week==		==Second Week==

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Revision as of 11:05, 27 June 2011

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Contents 1 Protocols 2 First Week 3 Second Week 4 Third Week 5 Fourth Week

Protocols

First Week

Second Week

Third Week

Fourth Week

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