Team:Copenhagen/Notebook

From 2011.igem.org

(Difference between revisions)
(→Notebook)
(→To Do - Future Plans)
 
(14 intermediate revisions not shown)
Line 1: Line 1:
-
{|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
+
-
 
+
-
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==
+
-
 
+
-
==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.
+

Latest revision as of 20:05, 21 September 2011

June
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
July
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
August
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31
September
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30
October
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
Retrieved from "http://2011.igem.org/Team:Copenhagen/Notebook"