Team:Copenhagen/Notebook

From 2011.igem.org

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

Revision as of 11:05, 27 June 2011

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Contents

Protocols

First Week

Second Week

Third Week

Fourth Week

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.