Team:TPHS SanDiego/Protocol

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Revision as of 01:03, 17 May 2013

Put protocol here!



CPEC
Wobble
EIPCR (enzymatic inverse PCR)
Normal ligation assembly (3A assembly)
Mini-preps
1) Resuspend pelleted bacterial cells in 250 µl (320 µL) Resuspension Buffer and transfer to a microcentrifuge tube.
2) Add 250 µl (320 µL) Lysis Buffer and mix by gentle inversion the tube 4–6 times to mix. Do not vortex, as this will result in shearing of genomic DNA
3) Add 350 µl (450 µL) Neutralization Buffer and invert the tube immediately but gently 4–6 times.
4) Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge.
5) Transfer the supernatant in a clean Eppendorf tube.
6) Add 0.6 volumes of isopropanol.
7) Centrifuge 10 minutes at maximum speed in a table top centrifuge
8) Wash with 70% ethanol.
9) Air dry and resuspend in 10 mM Tris pH = 7.4 (or ddH 2O)


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