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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>
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 |
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
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