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05/09/13
From 2013.igem.org
(Difference between revisions)
(→Double digest of isolated plasmid) |
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| Line 52: | Line 52: | ||
*1.2 ul of XbaI | *1.2 ul of XbaI | ||
*2.4 of SpeI | *2.4 of SpeI | ||
| - | 44.4 ul of 5mTris Hcl | + | *44.4 ul of 5mTris Hcl |
| + | |||
| + | Protocol | ||
| + | |||
| + | {|border=1 | ||
| + | |Sample||DNA (ul)||Master mix (ul)||5mTris Hcl (ul) added to final volume of 30ul | ||
| + | |- | ||
| + | |TodX 1||13.2 ||9 ||2.02 ||7.8 | ||
| + | |- | ||
| + | |TodX 2||8.5 ||9 ||1.9 ||12.5 | ||
| + | |- | ||
| + | |TodF 1||8.5 ||9 ||1.9||19 | ||
| + | |- | ||
| + | |TodF 2||5||9||1.92||2 | ||
| + | |- | ||
| + | |ToBG 1||9.5 ||9 ||1.94||11.6 | ||
| + | |- | ||
| + | |ToBG 2||23||9||2.00||0 | ||
| + | |} | ||
Revision as of 09:16, 6 September 2013
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Transformation of pSB1C3/TOD genes
- Centrifuge the ligation
- Add 6 ul of each ligation reaction to a sterile 1.5ml tube on ice
- Thaw the cells on ice
- Carefully transfer 50ul of cells to the ligation reaction. Gently flick the tubes and incubate on ice for 20 minutes.
- Heat shock the cells for 45 seconds in a 42'C water bath. Do not shake. Immediately return the tubes to ice for 2 minutes.
- Add 950 ul of SOC medium (at room temperature) to the ligation reactions. Incubate for 1.5 hours at 37'C on a shaker.
The controls for this experiment are the resistance and viability controls. One tube has 50 ul of highly competent cells and SOC medium added to it.
The samples A (TodX), C (TodF), E(ToBG), positive control, and background control were plated on chlorophenicol resistant agar plates, at the volumes of 20ul and 200ul. A 100ul of only highly efficient cells and SOC medium was plated in a chlorophenicol plate and another 100ul was plated on a LB plate.
Isolate plasmid from overnight broth
The plasmids were isolated using the Omega Plasmid Mini Kit 1, and its protocol was followed.
Nanodrop results
| Sample | Volume | Concentration ng/ul | 260/280 | 260/230 |
| TodX 1 | 39 | 37.8 | 2.02 | 1.72 |
| TodX 2 | 43.8 | 59.1 | 1.9 | 1.74 |
| TodF 1 | 42 | 100.4 | 1.9 | 2.02 |
| TodF 2 | 40.5 | 26.2 | 1.92 | 1.66 |
| ToBG 1 | 45 | 53.2 | 1.94 | 1.67 |
| ToBG 2 | 47 | 21.6 | 2.00 | 1.43 |
Double digest of isolated plasmid with SpeI and XbaI
Master mix for 12 reactions
- 36 ul of cut smart buffer
- 1.2 ul of XbaI
- 2.4 of SpeI
- 44.4 ul of 5mTris Hcl
Protocol
| Sample | DNA (ul) | Master mix (ul) | 5mTris Hcl (ul) added to final volume of 30ul | |
| TodX 1 | 13.2 | 9 | 2.02 | 7.8 |
| TodX 2 | 8.5 | 9 | 1.9 | 12.5 |
| TodF 1 | 8.5 | 9 | 1.9 | 19 |
| TodF 2 | 5 | 9 | 1.92 | 2 |
| ToBG 1 | 9.5 | 9 | 1.94 | 11.6 |
| ToBG 2 | 23 | 9 | 2.00 | 0 |
