Team:Lethbridge/Lab Work

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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 37^oC (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

<a name="may"></a> 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</td>	Buffer</td>	Volume MM(µL)</td>	Volume Enzyme(µL)</td></tr>
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</td>	per tube(µL)</td>	Total*(µL)</td></tr>
MilliQ H₂0	15.75	86.675
Red Buffer (10x)	2	11
pDNA**	2	11

Tango MM</td>	per tube(µL)</td>	Total*(µL)</td></tr>
MilliQ H₂0	15.75	86.675
Tango Buffer (10x)	2	11
pDNA**	2	11

Volume per reaction multiplied by 5.5
- Unknown concentration of pDNA

Incubated for 70min at 37^oC (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</td>	Sample</td>	Volume Loaded (µL)</td></tr>
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	Ladder	4

Ran gel at 100V for 1 hour

Results:

This gel shows that SpeI does not cut on its own, and does not cut when combined with other enzymes
Conclusion: Test other source of SpeI to see if it has any activity.

May 6/2010(in the lab:KG, AS)
Objective: To check if the old SpeI enzyme (exp date: March 2011) will cleave plasmid DNA, since we believe the newer SpeI enzyme (exp date: 2012) does not.
Method:

Red Master Mix	per tube (µL)	Total Volume*
MilliQ H₂0 Water	15.75	63
Red Buffer (10x)	2	8
pDNA**	2	8

Volume per tube multiplied by 4
- Used pSB NEYFP pDNA from cell E5 in plasmid box

Enzymes that will use Red Master Mix are: EcoRI+SpeI (old), EcoRI+SpeI (new)
Add 0.25µL of each enzyme to 19.5µL of master mix

Tango Master Mix	per tube (µL)	Total Volume*
MilliQ H₂0 Water	15.75	94.5
Tango Buffer (10x)	2	12
pDNA**	2	12

Volume per tube multiplied by 6
- Used pSB NEYFP pDNA from cell E5 in plasmid box

Enzymes that will use Tango Master Mix are: SpeI (old), SpeI (new), XbaI+SpeI (old), XbaI+SpeI (new)
Add 0.25µL of each enzyme to 19.5µL of master mix

Incubated all reactions at 37^oC for 1h (Start-8:30pm; End-9:30pm)
Will not be able to run on agarose gel tonight, will label them so JV can run them in the morning
Tube Names:
Master Mix 1 Control (Red Buffer)
Master Mix 2 Control (Tango Buffer)
E+S(N); EcoRI + SpeI(N)
E+S(O); EcoRI + SpeI(O)
X+S(N); XbaI + SpeI(N)
X+S(O); XbaI + SpeI(O)
S(N); SpeI(N)
S(O); SpeI(O)

Placed in -20^oC freezer of later analysis by agarose electrophoresis

May 10/2010(in the lab:JV)
Objective: To analyze the restriction test done by KG and AS on May 6/2010 by agarose electrophoresis

Method:

Lane	Sample	Quantity Loaded (µL)
1	MM1 Control	10
2	MM2 Control	10
3	EcoRI+SpeI(N)	10
4	EcoRI+SpeI(O)	10
5	SpeI(N)	10
6	SpeI(O)	10
7	XbaI+SpeI(N)	10
8	XbaI+SpeI(O)	10
9	1kb Ladder	5

Run gel for 60min at 100V

Results:

It appears as though both SpeI enzymes are working properly here. We will utilize the newer batch of SpeI (expires 2012) from this point forward.

@@ Line 1: / Line 1: @@
-This is our lab work page
+<b><font size=+2>April 13/2010</font> (In the Lab: JV, AS)</b><br>
+<b>Objective:</b> Test Restriction Endonucleases for Activity<br>
+<b>Relevant Information:</b><br>
+Endonucleases available
+<table><table border="3">
+<tr><td>Endonuclease</td><td>Optimal Buffer**</td><td>Other Buffers</td></tr>
+<tr><td>EcoRV</td><td>None</td><td>2xT(100%); O,G(50-100%)</td></tr>
+<tr><td>EcoRI</td><td>Red</td><td>O(100%);R(100%)*;2xT(100%)</td></tr>
+<tr><td>BcuI/SpeI</td><td>Tango</td><td>B(50-100%);G(50-100%)</td></tr>
+<tr><td>XbaI</td><td>Tango</td><td>B,G,2xT(50-100%)</td></tr>
+<tr><td>PstI</td><td>Orange</td><td>R(100%); B,G,T,2xT(50-100%)</td></tr>
+<tr><td>DpnI</td><td>Tango</td><td>B,G(100%): O,R,2xT(50-100%)</td></tr>
+</table>
+*Star Activity<br>
+**Optimal Buffer from Fermentas<br><br>
+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 <br>
+<u>Red Buffer:</u> EcoRI, PstI, Control (No Enzyme)<br>
+<u>Tango Buffer:</u> BcuI/SpeI, XbaI, DpnI, Control (No Enzyme><br><br>
+<u>Methods:</u>
+Set up Master Mixes:
+<table><table border="3">
+<tr><td><b>Red MM</b></td><td>per tube (&micro;L)</td><td>Total (&micro;L)</td></tr>
+<tr><td>MilliQ H<sub>2</sub>0</td><td>13.75</td><td>55</td></tr>
+<tr><td>Red Buffer (10x)</td><td>2</td><td>7</td></tr>
+<tr><td>pUC19 (10pg/&micro;L)</td><td>2</td><td>7</td></tr>
+<tr><td><b>Total</b></td><td>19.75</td><td>69</td></tr>
+</table><br>
+<table><table border="3">
+<tr><td><b>Tango MM</b></td><td>per tube (&micro;L)</td><td>Total (&micro;L)</td></tr>
+<tr><td>MilliQ H<sub>2</sub>0</td><td>13.75</td><td>55</td></tr>
+<tr><td>Tango Buffer (10x)</td><td>2</td><td>7</td></tr>
+<tr><td>pUC19 (10pg/&micro;L)</td><td>2</td><td>7</td></tr>
+<tr><td><b>Total</b></td><td>19.75</td><td>69</td></tr>
+</table><br>
+To each tube, add <b>19.75&micro;L</b> of master mix and <b>0.25&micro;L</b> of enzyme<br>
+Incubated reaction mixes at 37<sup>o</sup>C (Start:7:00pm; End:7:45pm)<br>
+Add 3.3&micro;L of 6x loading dye to each reaction mixture and load 10&micro;L final volume onto a 1% agarose (in TAE) gel.<br>
+Add 1&micro;L of 6x loading dye to 1&micro;L of GeneRuler 1kb ladder (at 0.5&micro;g/&micro;L)<br>
+Gel loading order as follows:<br>
+<table><table border="3">
+<tr><td><b>Lane</b></td><td><b>Sample</b></td></tr>
+<tr><td>1</td><td>1kb Ladder</td></tr>
+<tr><td>2</td><td>Tango Control</td></tr>
+<tr><td>3</td><td>DpnI (Tango)</td></tr>
+<tr><td>4</td><td>BcuI/SpeI (Tango)</td></tr>
+<tr><td>5</td><td>XbaI (Tango)</td></tr>
+<tr><td>6</td><td>EcoRI (Red)</td></tr>
+<tr><td>7</td><td>PstI (Red)</td></tr>
+<tr><td>8</td><td>Red Control</td></tr>
+<tr><td>9</td><td>Empty</td></tr>
+<tr><td>10</td><td>Empty</td></tr>
+</table><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>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>
+<a name="may"></a>
+<b><font size=+2>May 5/2010</font>(in the lab: JV)</b><br>
+<b>Objective:</b> Test Restriction Endonucleases for activity (take 2)<br>
+<b>Relevant Information:</b><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>
+*Volume per reaction multiplied by 5.5<br>
+**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>Ladder</td><td>4</td></tr>
+</table><br>
+Ran gel at 100V for 1 hour<br><br>
+<b>Results:</b><br>
+[[image:100505JV-EnzymeTest1Cropped.jpg|200px|none]]
+This gel shows that SpeI does not cut on its own, and does not cut when combined with other enzymes<br>
+<b>Conclusion:</b> Test other source of SpeI to see if it has any activity.<br><br>
+<b><font size=+2>May 6/2010</font>(in the lab:KG, AS)</b><br>
+<b>Objective:</b> To check if the old SpeI enzyme (exp date: March 2011) will cleave plasmid DNA, since we believe the newer SpeI enzyme (exp date: 2012) does not.<br>
+<b>Method:</b><br>
+<table><table border ="3">
+<tr><td><b>Red Master Mix</b></td><td>per tube (&micro;L)</td><td>Total Volume*</td></tr>
+<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.75</td><td>63</td></tr>
+<tr><td>Red Buffer (10x)</td><td>2</td><td>8</td></tr>
+<tr><td>pDNA**</td><td>2</td><td>8</td></tr>
+</table>
+*Volume per tube multiplied by 4<br>
+**Used pSB NEYFP pDNA from cell E5 in plasmid box<br>
+Enzymes that will use Red Master Mix are: EcoRI+SpeI (old), EcoRI+SpeI (new)<br>
+Add 0.25&micro;L of each enzyme to 19.5&micro;L of master mix<br><br>
+<table><table border ="3">
+<tr><td><b>Tango Master Mix</b></td><td>per tube (&micro;L)</td><td>Total Volume*</td></tr>
+<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>15.75</td><td>94.5</td></tr>
+<tr><td>Tango Buffer (10x)</td><td>2</td><td>12</td></tr>
+<tr><td>pDNA**</td><td>2</td><td>12</td></tr>
+</table>
+*Volume per tube multiplied by 6<br>
+**Used pSB NEYFP pDNA from cell E5 in plasmid box<br>
+Enzymes that will use Tango Master Mix are: SpeI (old), SpeI (new), XbaI+SpeI (old), XbaI+SpeI (new)<br>
+Add 0.25&micro;L of each enzyme to 19.5&micro;L of master mix<br><br>
+Incubated all reactions at 37<sup>o</sup>C for 1h (Start-8:30pm; End-9:30pm)<br>
+Will not be able to run on agarose gel tonight, will label them so JV can run them in the morning<br>
+<u>Tube Names:</u><br>
+Master Mix 1 Control (Red Buffer)<br>
+Master Mix 2 Control (Tango Buffer)<br>
+E+S(N); EcoRI + SpeI(N)<br>
+E+S(O); EcoRI + SpeI(O)<br>
+X+S(N); XbaI + SpeI(N)<br>
+X+S(O); XbaI + SpeI(O)<br>
+S(N); SpeI(N)<br>
+S(O); SpeI(O)<br><br>
+Placed in -20<sup>o</sup>C freezer of later analysis by agarose electrophoresis<br><br>
+<b><font size=+2>May 10/2010</font>(in the lab:JV)</b><br>
+<b>Objective:</b> To analyze the restriction test done by KG and AS on May 6/2010 by agarose electrophoresis<br><br>
+<b>Method:</b><br>
+<table><table border="3">
+<tr><td><b>Lane</b></td><td>Sample</td><td>Quantity Loaded (&micro;L)</td></tr>
+<tr><td>1</td><td>MM1 Control</td><td>10</td></tr>
+<tr><td>2</td><td>MM2 Control</td><td>10</td></tr>
+<tr><td>3</td><td>EcoRI+SpeI(N)</td><td>10</td></tr>
+<tr><td>4</td><td>EcoRI+SpeI(O)</td><td>10</td></tr>
+<tr><td>5</td><td>SpeI(N)</td><td>10</td></tr>
+<tr><td>6</td><td>SpeI(O)</td><td>10</td></tr>
+<tr><td>7</td><td>XbaI+SpeI(N)</td><td>10</td></tr>
+<tr><td>8</td><td>XbaI+SpeI(O)</td><td>10</td></tr>
+<tr><td>9</td><td>1kb Ladder</td><td>5</td></tr>
+</table>
+Run gel for 60min at 100V<br><br>
+<b>Results:</b><br>
+[[image:100510JRV-EnzymeTest1Cropped.jpg|200px|none]]<br>
+It appears as though both SpeI enzymes are working properly here. We will utilize the newer batch of SpeI (expires 2012) from this point forward.<br><br>