Team:Lethbridge

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April 13/2010 (In the Lab: JV, AS)
Objective: Test Restriction Endonucleases for Activity
Related 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

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:

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