This is where our electronic lab book data will go, gel pictures will also go here and here is an example of it:<br>
This is where our electronic lab book data will go, gel pictures will also go here and here is an example of it:<br>
Revision as of 04:44, 14 May 2010
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University of Lethbridge IGEM team
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April 13/2010 (In the Lab: JV, AS) Objective: Test Restriction Endonucleases for Activity Relevant Information:
Endonucleases available
Endonuclease
Optimal Buffer**
Other Buffers
EcoRV
None
2xT(100%); O,G(50-100%)
EcoRI
Red
O(100%);R(100%)*;2xT(100%)
BcuI/SpeI
Tango
B(50-100%);G(50-100%)
XbaI
Tango
B,G,2xT(50-100%)
PstI
Orange
R(100%); B,G,T,2xT(50-100%)
DpnI
Tango
B,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 MM
per tube (µL)
Total (µL)
MilliQ H20
13.75
55
Red Buffer (10x)
2
7
pUC19 (10pg/µL)
2
7
Total
19.75
69
Tango MM
per tube (µL)
Total (µL)
MilliQ H20
13.75
55
Tango Buffer (10x)
2
7
pUC19 (10pg/µL)
2
7
Total
19.75
69
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:
Lane
Sample
1
1kb Ladder
2
Tango Control
3
DpnI (Tango)
4
BcuI/SpeI (Tango)
5
XbaI (Tango)
6
EcoRI (Red)
7
PstI (Red)
8
Red Control
9
Empty
10
Empty
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:
Enzyme
Buffer
Volume MM(µL)
Volume Enzyme(µL)
PstI
Red
19.75
.25
XbaI
Tango
19.75
.25
SpeI
Tango
19.75
.25
EcoRI
Red
19.75
.25
EcoRI/SpeI
Red
19.5
.25+.25
XbaI/SpeI
Tango
19.5
.25+.25
EcoRI/PstI
Red
19.5
.25+.25
XbaI/PstI
Tango
19.5
.25+.25
Make up Master Mixes as follows:
Red MM
per tube(µL)
Total*(µL)
MilliQ H20
15.75
86.675
Red Buffer (10x)
2
11
pDNA**
2
11
Tango MM
per tube(µL)
Total*(µL)
MilliQ H20
15.75
86.675
Tango Buffer (10x)
2
11
pDNA**
2
11
*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:
Lane
Sample
Volume Loaded (µL)
1
pSB-CEYFP/PstI
10
2
pSB-CEYFP/EcoRI
10
3
pSB-CEYFP/EcoRI/PstI
10
4
pSB-CEYFP/EcoRI/SpeI
10
5
pSB-CEYFP/XbaI/PstI
10
6
pSB-CEYFP/XbaI
10
7
pSB-CEYFP/SpeI
10
8
pSB-CEYFP/XbaI/SpeI
10
9
pSB-CEYFP/Red Master Mix Control
10
10
pSB-CEYFP/Tango Master Mix Control
10
11
pSB-CEYFP/MilliQ H20 Control
10
12
pSB-CEYFP/Ladder
10
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: