Team:Lethbridge

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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


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 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	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:

Content 6

Content 7