Team:Lethbridge

From 2010.igem.org

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<b><font=+1>April 13/2010 (In the Lab: JV, AS)</font></b><br>
+
<b><font size=+2>April 13/2010</font> (In the Lab: JV, AS)</b><br>
<u>Objective:</u> Test Restriction Endonucleases for Activity<br>
<u>Objective:</u> Test Restriction Endonucleases for Activity<br>
-
<u>Related Information:</u><br>
+
<u>Relevant Information:</u><br>
Endonucleases available
Endonucleases available
<table><table border="3">
<table><table border="3">
Line 113: Line 113:
Ran gel at 100V for 1 hour<br>
Ran gel at 100V for 1 hour<br>
<b>Results:</b> pUC19 plasmid DNA not present at a high enough concentration to visualize by ethidium bromide staining (1kb ladder did stain). <br>
<b>Results:</b> pUC19 plasmid DNA not present at a high enough concentration to visualize by ethidium bromide staining (1kb ladder did stain). <br>
-
<b>Conclusion:</b> Will have to re-run experiment with DNA that is present at high enough concentrations to visualize by ethidium bromide staining<br>
+
<b>Conclusion:</b> Will have to re-run experiment with DNA that is present at high enough concentrations to visualize by ethidium bromide staining<br><br><br>
 +
<b><font size=+2>May 5/2010</font>(in the lab: JV)</b><br>
 +
<u>Objective:</u> Test Restriction Endonucleases for activity (take 2)<br>
 +
<u>Relevant Information:</u><br>
 +
Plasmid DNA used here will be "ES-pSB-CEYFP" from last year's plasmid stocks<br>
 +
Prefix Enzymes are: EcoRI and XbaI<br>
 +
Suffix Enyzmes are: SpeI and PstI<br>
 +
(JV worked out in lab notebook which buffers would be best for each prefix/suffix enzyme combination)<br>
 +
Reactions will be assembled as follows:<br>
 +
<table><table border="3">
 +
<tr><td><b>Enzyme</td><td>Buffer</td><td>Volume MM(&micro;L)</td><td>Volume Enzyme(&micro;L)</td></tr></b>
 +
<tr><td>PstI</td><td>Red</td><td>19.75</td><td>.25</td></tr>
 +
<tr><td>XbaI</td><td>Tango</td><td>19.75</td><td>.25</td></tr>
 +
<tr><td>SpeI</td><td>Tango</td><td>19.75</td><td>.25</td></tr>
 +
<tr><td>EcoRI</td><td>Red</td><td>19.75</td><td>.25</td></tr>
 +
<tr><td>EcoRI/SpeI</td><td>Red</td><td>19.5</td><td>.25+.25</td></tr>
 +
<tr><td>XbaI/SpeI</td><td>Tango</td><td>19.5</td><td>.25+.25</td></tr>
 +
<tr><td>EcoRI/PstI</td><td>Red</td><td>19.5</td><td>.25+.25</td></tr>
 +
<tr><td>XbaI/PstI</td><td>Tango</td><td>19.5</td><td>.25+.25</td></tr>
 +
</table><br>
 +
Make up Master Mixes as follows:<br>
 +
<table><table border="3">
 +
<tr><td><b>Red MM</td><td>per tube(&micro;L)</td><td>Total*(&micro;L)</td></tr></b>
 +
<tr><td>MilliQ H<sub>2</sub>0</td><td>15.75</td><td>86.675</td></tr>
 +
<tr><td>Red Buffer (10x)</td><td>2</td><td>11</td></tr>
 +
<tr><td>pDNA**</td><td>2</td><td>11</td></tr>
 +
</table><br>
 +
<table><table border="3">
 +
<tr><td><b>Tango MM</td><td>per tube(&micro;L)</td><td>Total*(&micro;L)</td></tr></b>
 +
<tr><td>MilliQ H<sub>2</sub>0</td><td>15.75</td><td>86.675</td></tr>
 +
<tr><td>Tango Buffer (10x)</td><td>2</td><td>11</td></tr>
 +
<tr><td>pDNA**</td><td>2</td><td>11</td></tr>
 +
</table><br>
 +
*Volume per reaction time 5.5
 +
**Unknown concentration of pDNA<br><br>
 +
Incubated for 70min at 37<sup>o</sup>C (Start-1:05pm; End-2:15pm)<br>
 +
Added 3.3&micro;L of 6x loading dye to each reaction mixture and loaded 10&micro;L onto a 1% agarose gel (in TAE)<br>
 +
Added 1&micro;L of 6x loading dye to 2&micro;L of gene ruler 1kb ladder<br>
 +
Load order as follows:<br>
 +
<table><table border="3">
 +
<tr><td><b>Lane</td><td>Sample</td><td>Volume Loaded (&micro;L)</td></tr></b>
 +
<tr><td>1</td><td>pSB-CEYFP/PstI</td><td>10</td></tr>
 +
<tr><td>2</td><td>pSB-CEYFP/EcoRI</td><td>10</td></tr>
 +
<tr><td>3</td><td>pSB-CEYFP/EcoRI/PstI</td><td>10</td></tr>
 +
<tr><td>4</td><td>pSB-CEYFP/EcoRI/SpeI</td><td>10</td></tr>
 +
<tr><td>5</td><td>pSB-CEYFP/XbaI/PstI</td><td>10</td></tr>
 +
<tr><td>6</td><td>pSB-CEYFP/XbaI</td><td>10</td></tr>
 +
<tr><td>7</td><td>pSB-CEYFP/SpeI</td><td>10</td></tr>
 +
<tr><td>8</td><td>pSB-CEYFP/XbaI/SpeI</td><td>10</td></tr>
 +
<tr><td>9</td><td>pSB-CEYFP/Red Master Mix Control</td><td>10</td></tr>
 +
<tr><td>10</td><td>pSB-CEYFP/Tango Master Mix Control</td><td>10</td></tr>
 +
<tr><td>11</td><td>pSB-CEYFP/MilliQ H<sub>2</sub>0 Control</td><td>10</td></tr>
 +
<tr><td>12</td><td>pSB-CEYFP/Ladder</td><td>10</td></tr>
 +
</table><br>
 +
Ran gel at 100V for 1 hour<br><br>
 +
<b>Results:</b><br>
 +
 
 +
 

Revision as of 04:27, 14 May 2010

Untitled Document

University of Lethbridge IGEM team

 

  • Main
  • Team
  • Projects
  • Notebook
  • Meetings
  • Parts
  • Collaborations
blah balh balh

Our team is based yada yada

  • Team Pics
  • Tab 3
  • Team bios
Team pictures
Content 3
Pics of us

 

 

 

 

 

Overview

  • Project 1
  • Porejct 2
Content 1
Content 2

 

 

 

April 13/2010 (In the Lab: JV, AS)
Objective: Test Restriction Endonucleases for Activity
Relevant Information:
Endonucleases available
EndonucleaseOptimal Buffer**Other Buffers
EcoRVNone2xT(100%); O,G(50-100%)
EcoRIRedO(100%);R(100%)*;2xT(100%)
BcuI/SpeITangoB(50-100%);G(50-100%)
XbaITangoB,G,2xT(50-100%)
PstIOrangeR(100%); B,G,T,2xT(50-100%)
DpnITangoB,G(100%): O,R,2xT(50-100%)
*Star Activity
**Optimal Buffer from Fermentas

Use pUC19 plasmid as test, it has cut sites for EcoRI, PstI, XbaI (unsure about BcuI/SpeI, DpnI but will try anyways), and none for EcoRV
Red Buffer: EcoRI, PstI, Control (No Enzyme)
Tango Buffer: BcuI/SpeI, XbaI, DpnI, Control (No Enzyme>

Methods: Set up Master Mixes:
Red MMper tube (µL)Total (µL)
MilliQ H2013.7555
Red Buffer (10x)27
pUC19 (10pg/µL)27
Total19.7569

Tango MMper tube (µL)Total (µL)
MilliQ H2013.7555
Tango Buffer (10x)27
pUC19 (10pg/µL)27
Total19.7569

To each tube, add 19.75µL of master mix and 0.25µL of enzyme
Incubated reaction mixes at 37oC (Start:7:00pm; End:7:45pm)
Add 3.3µL of 6x loading dye to each reaction mixture and load 10µL final volume onto a 1% agarose (in TAE) gel.
Add 1µL of 6x loading dye to 1µL of GeneRuler 1kb ladder (at 0.5µg/µL)
Gel loading order as follows:
LaneSample
11kb Ladder
2Tango Control
3DpnI (Tango)
4BcuI/SpeI (Tango)
5XbaI (Tango)
6EcoRI (Red)
7PstI (Red)
8Red Control
9Empty
10Empty

Ran gel at 100V for 1 hour
Results: pUC19 plasmid DNA not present at a high enough concentration to visualize by ethidium bromide staining (1kb ladder did stain).
Conclusion: Will have to re-run experiment with DNA that is present at high enough concentrations to visualize by ethidium bromide staining


May 5/2010(in the lab: JV)
Objective: Test Restriction Endonucleases for activity (take 2)
Relevant Information:
Plasmid DNA used here will be "ES-pSB-CEYFP" from last year's plasmid stocks
Prefix Enzymes are: EcoRI and XbaI
Suffix Enyzmes are: SpeI and PstI
(JV worked out in lab notebook which buffers would be best for each prefix/suffix enzyme combination)
Reactions will be assembled as follows:
EnzymeBufferVolume MM(µL)Volume Enzyme(µL)
PstIRed19.75.25
XbaITango19.75.25
SpeITango19.75.25
EcoRIRed19.75.25
EcoRI/SpeIRed19.5.25+.25
XbaI/SpeITango19.5.25+.25
EcoRI/PstIRed19.5.25+.25
XbaI/PstITango19.5.25+.25

Make up Master Mixes as follows:
Red MMper tube(µL)Total*(µL)
MilliQ H2015.7586.675
Red Buffer (10x)211
pDNA**211

Tango MMper tube(µL)Total*(µL)
MilliQ H2015.7586.675
Tango Buffer (10x)211
pDNA**211

*Volume per reaction time 5.5 **Unknown concentration of pDNA

Incubated for 70min at 37oC (Start-1:05pm; End-2:15pm)
Added 3.3µL of 6x loading dye to each reaction mixture and loaded 10µL onto a 1% agarose gel (in TAE)
Added 1µL of 6x loading dye to 2µL of gene ruler 1kb ladder
Load order as follows:
LaneSampleVolume Loaded (µL)
1pSB-CEYFP/PstI10
2pSB-CEYFP/EcoRI10
3pSB-CEYFP/EcoRI/PstI10
4pSB-CEYFP/EcoRI/SpeI10
5pSB-CEYFP/XbaI/PstI10
6pSB-CEYFP/XbaI10
7pSB-CEYFP/SpeI10
8pSB-CEYFP/XbaI/SpeI10
9pSB-CEYFP/Red Master Mix Control10
10pSB-CEYFP/Tango Master Mix Control10
11pSB-CEYFP/MilliQ H20 Control10
12pSB-CEYFP/Ladder10

Ran gel at 100V for 1 hour

Results:

May 13
This is what we did today
This is where our electronic lab book data will go, gel pictures will also go here and here is an example of it:
Content 6
Content 7

 

 

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