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| - | {|align="justify" | + | {{:Team:Copenhagen/Header}} |
| - | |You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing.
| + | <html> |
| - | |[[Image:Copenhagen_logo.png|200px|right|frame]]
| + | <head> |
| - | |-
| + | <style> |
| - | |
| + | table.calendar td { |
| - | ''Tell us more about your project. Give us background. Use this is the abstract of your project. Be descriptive but concise (1-2 paragraphs)''
| + | vertical-align:top; |
| - | |[[Image:Copenhagen_team.png|right|frame|Your team picture]]
| + | align:center; |
| - | |-
| + | } |
| - | |
| + | |
| - | |align="center"|[[Team:Copenhagen | Team Example]]
| + | |
| - | |}
| + | |
| | | | |
| - | <!--- The Mission, Experiments --->
| + | table.month { |
| | + | background-color:#FFF; |
| | + | border:solid 1px; |
| | + | } |
| | | | |
| - | {| style="color:#3b3c3a;background-color:#0c11;" cellpadding="6" cellspacing="2" border="3" bordercolor="#fff" width="82%" align="center" | + | table.month .heading td { |
| - | !align="center"|[[Team:Copenhagen|Home]]
| + | background-color:#800080; |
| - | !align="center"|[[Team:Copenhagen/Team|Team]]
| + | color:#FFF; |
| - | !align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Copenhagen Official Team Profile]
| + | text-align:center; |
| - | !align="center"|[[Team:Copenhagen/Project|Project]]
| + | font-weight:bold; |
| - | !align="center"|[[Team:Copenhagen/Parts|Parts Submitted to the Registry]]
| + | text-size:1200px; |
| - | !align="center"|[[Team:Copenhagen/Notebook|Notebook]]
| + | } |
| - | !align="center"|[[Team:Copenhagen/Safety|Safety]]
| + | |
| - | !align="center"|[[Team:Copenhagen/Attributions|Attributions]]
| + | |
| - | |}
| + | |
| | | | |
| | + | table.month .dow td { |
| | + | color:#000; |
| | + | text-align:center; |
| | + | font-size:100%; |
| | + | } |
| | | | |
| - | =='''Protocols'''==
| + | table.month td { |
| | + | border:none; |
| | + | text-align:center; |
| | + | background-color:#FFF; |
| | + | } |
| | | | |
| - | ==Protocols==
| + | table.month a:hover { |
| | + | background-color:#800080; |
| | + | text-decoration:none; |
| | + | } |
| | | | |
| - | ===Mutations===
| + | .day-active { |
| | + | color:red; |
| | + | font-weight:bold; |
| | + | } |
| | | | |
| - | '''Ingredients'''
| + | .day-empty { |
| | + | color:#000; |
| | | | |
| - | Site directed mutagenesis of 3 plant CYP450 (79A1, 79A2, 79B1).
| + | } |
| | + | </style> |
| | + | </head> |
| | + | </html> |
| | | | |
| - | We aim to destroy restrictionenzymes recognitionsites.
| + | {|align="center" |
| - | | + | |- |
| - | Primers was supplied by ...........
| + | |width="20%"align="center" valign="top"|{{ #calendar: title=Copenhagen|year=2011| month=06 }} |
| - | | + | |width="20%"align="center" valign="top"|{{ #calendar: title=Copenhagen|year=2011| month=07 }} |
| - | | + | |width="20%"align="center" valign="top"|{{ #calendar: title=Copenhagen|year=2011| month=08 }} |
| - | 10 μl of 5× reaction buffer
| + | |width="20%"align="center" valign="top"|{{ #calendar: title=Copenhagen|year=2011| month=09 }} |
| - | | + | |width="20%"align="center" valign="top"|{{ #calendar: title=Copenhagen|year=2011| month=10 }} |
| - | X μl (50 ng) of dsDNA template
| + | |} |
| - | | + | |
| - | X μl (125 ng) of oligonucleotide primer #1
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| - | | + | |
| - | 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
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| - | | + | |
| - | 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
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| - | 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== | + | |
| - | | + | |
| - | ==Third Week== | + | |
| - | | + | |
| - | ==Fourth Week== | + | |
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| - | 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.
| + | |
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