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<BLOCKQUOTE>
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Back to [[Team:Lethbridge/Notebook|Notebook]]<br>
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=<font color="white">April=
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Back to [[Team:Lethbridge/Notebook/Lab_Work|Lab Work]]
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==<font color="white">April 13/2010==
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=April=
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==April 13/2010==
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(In the Lab: JV, AS)<br>
(In the Lab: JV, AS)<br>
<b>Objective:</b> Test Restriction Endonucleases for Activity<br>
<b>Objective:</b> Test Restriction Endonucleases for Activity<br>
Line 63: Line 206:
Endonucleases available
Endonucleases available
<table><table border="3">
<table><table border="3">
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<tr><td>Endonuclease</td><td>Optimal Buffer(**)</td><td>Other Buffers</td></tr>
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<tr><td><b>Endonuclease</b></td><td><b>Optimal Buffer(**)</b></td><td><b>Other Buffers</b></td></tr>
<tr><td>EcoRV</td><td>None</td><td>2xT(100%); O,G(50-100%)</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>EcoRI</td><td>Red</td><td>O(100%);R(100%)(*);2xT(100%)</td></tr>
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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>
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>Red Buffer:</u> EcoRI, PstI, Control (No Enzyme)<br>
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<u>Tango Buffer:</u> BcuI/SpeI, XbaI, DpnI, Control (No Enzyme><br><br>
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<u>Tango Buffer:</u> BcuI/SpeI, XbaI, DpnI, Control (No Enzyme)<br><br>
<b>Methods:</b>
<b>Methods:</b>
Set up Master Mixes:
Set up Master Mixes:

Latest revision as of 00:44, 18 October 2010



Feel free to look around our notebook!

Here you can check out the work we have done in the lab, click on a month to take a look!

April

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


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