Difference between revisions of "Team:UPO-Sevilla/Protocols"
| Line 139: | Line 139: | ||
<!-- /. PAGE TITLE--> | <!-- /. PAGE TITLE--> | ||
| − | + | <p style="font-size:20px;background-color:#E0F2F7;text-align:center"> | |
| − | <p style="font-size:20px;background-color:#E0F2F7; | + | |
Plasmid DNA extraction | Plasmid DNA extraction | ||
</p> | </p> | ||
| Line 176: | Line 175: | ||
</div> | </div> | ||
| − | + | <p style="font-size:20px;background-color:#E0F2F7;text-align:center"> | |
| − | + | ||
DNA electrophoresis | DNA electrophoresis | ||
</p> | </p> | ||
| Line 234: | Line 232: | ||
</div> | </div> | ||
| − | + | <p style="font-size:20px;background-color:#E0F2F7;text-align:center"> | |
| − | <p style="font-size:20px;background-color:#E0F2F7; | + | |
Ligation | Ligation | ||
</p> | </p> | ||
| Line 263: | Line 260: | ||
</div> | </div> | ||
| − | + | <p style="font-size:20px;background-color:#E0F2F7;text-align:center"> | |
| − | + | ||
Generate competent cells | Generate competent cells | ||
</p> | </p> | ||
| Line 304: | Line 300: | ||
</div> | </div> | ||
| − | < | + | <p style="font-size:20px;background-color:#E0F2F7;text-align:center"> |
| − | + | ||
Heat-shock transformation | Heat-shock transformation | ||
</p> | </p> | ||
| Line 337: | Line 332: | ||
</div> | </div> | ||
| − | + | <p style="font-size:20px;background-color:#E0F2F7;text-align:center"> | |
| − | + | ||
Electrocompetent cells | Electrocompetent cells | ||
</p> | </p> | ||
| Line 359: | Line 353: | ||
</div> | </div> | ||
| − | + | <p style="font-size:20px;background-color:#E0F2F7;text-align:center"> | |
| − | + | ||
Electroporation and transposition | Electroporation and transposition | ||
</p> | </p> | ||
| Line 410: | Line 403: | ||
</div> | </div> | ||
| − | < | + | <p style="font-size:20px;background-color:#E0F2F7;text-align:center"> |
| − | + | ||
| − | + | ||
Triparental mating | Triparental mating | ||
</a> | </a> | ||
| Line 445: | Line 436: | ||
</div> | </div> | ||
| − | + | <p style="font-size:20px;background-color:#E0F2F7;text-align:center"> | |
| − | + | ||
PCR (iPfu<sup>®</sup>) | PCR (iPfu<sup>®</sup>) | ||
</p> | </p> | ||
| Line 463: | Line 453: | ||
</div> | </div> | ||
| − | < | + | <p style="font-size:20px;background-color:#E0F2F7;text-align:center"> |
| − | + | ||
Digestion (NEB enzymes) | Digestion (NEB enzymes) | ||
</a> | </a> | ||
| Line 480: | Line 469: | ||
</div> | </div> | ||
| − | + | <p style="font-size:20px;background-color:#E0F2F7;text-align:center"> | |
| − | + | ||
Site-directed mutagenesis using overlap extension PCR | Site-directed mutagenesis using overlap extension PCR | ||
</p> | </p> | ||
| Line 514: | Line 502: | ||
</div> | </div> | ||
| − | + | <p style="font-size:20px;background-color:#E0F2F7;text-align:center"> | |
| − | + | ||
Dilution series-based growth curves | Dilution series-based growth curves | ||
</p> | </p> | ||
| Line 575: | Line 562: | ||
</div> | </div> | ||
| − | + | <p style="font-size:20px;background-color:#E0F2F7;text-align:center"> | |
| − | + | ||
Beta-galactosidase assay | Beta-galactosidase assay | ||
</p> | </p> | ||
Revision as of 16:44, 4 October 2016
PROTOCOLS
Plasmid DNA extraction
-
Collect the saturated bacteria culture in a 1'5 mL tube and centrifuge at 13000 rpm for 1 minute. Do it twice
-
Add 100 μL of GTE (Tris HCl 25 mM, pH 8, glucose 50 mM, EDTA 10 mM), mix by inverting and incubate on ice for 5 minutes
-
Add 150 μL of potassium acetate 3 M (pH 4'8), mix by inverting and incubate on ice for at least 5 minutes
-
Centrifuge the tubes at 13000 rpm for 10 minutes and transfer the supernatant to a 1'5 mL tube
-
Add 400 μL of ice ethanol 96% and centrifuge at 13000 rpm for 10 minutes
-
Discard the supernatant, add 500 μL of ethanol 70% and centrifuge at 13000 rpm for 5 minutes
-
Let the tubes dry and resuspend the plasmid DNA in 50 μL of TER (TrisHCl 10 mM, pH 8, EDTA 1 mM, RNAse 20 mg L -1)
DNA electrophoresis
GEL MAKING
-
Prepare 40 or 100 mL of TAE (Trisacetate 40 mM, pH 7’7, EDTA 10 mM), it depends on the size of the gel
-
Add 0’4 g or 1 g of agarose to the TAE solution (for 1% gel)
-
Heat the solution until all the agarose has been dissolved and cold it to 50ºC
-
Pour the the solution in the electrophoresis vessel, apply the combs and let it polymerize
GEL RUNNING
-
Add 1 μL of loading buffer 6x (TrisHCl 5 mM, pH 8, EDTA 0’5 mM, glycerol 30%, xylene cyanol 2’5 g L -1, bromophenol blue 2’5 g L -1) to 5 μL of DNA sample
-
Remove the combs from the gel, put the vessel on a horizontal electrophoresis system and cover it with TAE
-
Pipette 6 μL of the DNA samples and ladder
-
Run at 120 mV for 45 minutes approximately
REVEAL THE GEL
-
Put the gel in ethidium bromide for 20 minutes
-
Look the gel on the transilluminator and take a photo of it
Ligation
-
Add 6 μL of insert DNA
-
Add 1 μL of vector DNA
-
Add 2 μL of ligase buffer high ATP (TrisHCl 50 mM, pH 7’6, MgCl 2 10 mM, DTT 5 mM, BSA 50 μg mL -1)
-
Add 1 μL of T4 DNA ligase
-
Incubate at 16ºC overnight
Generate competent cells
