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Team:TPHS SanDiego/Protocol
From 2013hs.igem.org
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<br> | <br> | ||
<br> | <br> | ||
| - | + | <br> <b> CPEC </b> | |
| - | + | <br> <b> Wobble </b> | |
| - | EIPCR (enzymatic inverse PCR) < | + | <br> <b> EIPCR (enzymatic inverse PCR) </b> |
| - | Normal ligation assembly (3A assembly) < | + | <br> <b> Normal ligation assembly (3A assembly) </b> |
| - | Mini-preps < | + | <br> Mini-preps </b> |
<br> | <br> | ||
<br> | <br> | ||
| - | <b> Transformations </b> | + | <b> <b> Transformations </b> |
<br> 1.Thaw competent cells on ice. | <br> 1.Thaw competent cells on ice. | ||
<br> 2.Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube. | <br> 2.Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube. | ||
| Line 73: | Line 73: | ||
<br> 10.Incubate overnight at 37°C | <br> 10.Incubate overnight at 37°C | ||
<br> * Please note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period, please follow the recommendations provided by the competent cells’ manufacturer. | <br> * Please note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period, please follow the recommendations provided by the competent cells’ manufacturer. | ||
| - | <br> <br> | + | <br> |
| - | + | <br> | |
| - | + | <br> <b> Ligations </b> | |
| - | + | <br> <b> Digestions </b> | |
| - | Normal PCRs < | + | <br> <b> Normal PCRs </b> |
| - | Gel-extractions < | + | <br> <b> Gel-extractions </b> |
| - | PCR-purifications < | + | <br> <b> PCR-purifications </b> |
| - | Running electrophoresis gels < | + | <br> <b> Running electrophoresis gels </b> |
| - | Comp cells < | + | <br> <b> Comp cells </b> |
| - | + | <br> <b> Plating </b> | |
| - | Overnight cultures (liquid cultures) < | + | <br> <b> Overnight cultures (liquid cultures) </b> |
<br> | <br> | ||
<br> | <br> | ||
Revision as of 00:56, 17 May 2013
Put protocol here!
CPEC
Wobble
EIPCR (enzymatic inverse PCR)
Normal ligation assembly (3A assembly)
Mini-preps
Transformations
1.Thaw competent cells on ice.
2.Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube.
3.Add 50 µl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA. Do not vortex.
4.Place the mixture on ice for 30 minutes. Do not mix.
5.Heat shock at 42°C for 30 seconds*. Do not mix.
6.Add 950 µl of room temperature media* to the tube.
7.Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8.Warm selection plates to 37°C.
9.Spread 50–100 µl of the cells and ligation mixture onto the plates.
10.Incubate overnight at 37°C
* Please note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period, please follow the recommendations provided by the competent cells’ manufacturer.
Ligations
Digestions
Normal PCRs
Gel-extractions
PCR-purifications
Running electrophoresis gels
Comp cells
Plating
Overnight cultures (liquid cultures)
Denote when the UCSD services had to be used – sequencing, designing primers, data managing techniques, ordering primers, thermocycler
