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| - | <!-- originale della home disponibile a https://2010.igem.org/Team:UNIPV-Pavia/homebackup -->
| + | <table width="100%" border="0" > |
| - | | + | |
| - | | + | |
| - | <table width="100%" border="0"> | + | |
| | <tr> | | <tr> |
| - | <td colspan="2"> {{UNIPV-Pavia/header}} </td>
| + | <td colspan="2">{{UNIPV-Pavia/header}}</td> |
| - | <!-- <td colspan="2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> -->
| + | |
| | </tr> | | </tr> |
| - | | + | <tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td><td style="padding:20px"> |
| - | <!-- <tr><td colspan="2"><html><marquee>countdown</marquee></html></td></tr> -->
| + | <p align="center" valign="top"><br> |
| - | | + | <font size="6" align="center" valign="top">'''''Material & Methods'''''</font> |
| - | <tr><td align="left" valign="top" width="20%">{{UNIPV-Pavia/menu}}</td> | + | </p> |
| - | | + | <hr> |
| - | <!-- Contenuti --> | + | |
| - | | + | |
| - | <td valign="top" > | + | |
| - | | + | |
| - | =Media & Antibiotics= | + | |
| - | | + | |
| - | ==LB== | + | |
| - | * Add:
| + | |
| - | ** 10 g/L NaCl
| + | |
| - | ** 10 g/L Bacto-Tryptone
| + | |
| - | ** 0.5 g/L Bacto-Yeast Extract
| + | |
| - | ** ddH2O
| + | |
| - | to a sterile pyrex bottle
| + | |
| - | * autoclave
| + | |
| - | * (add antibiotic when it reaches ~45°C)
| + | |
| - | * store at +4°C
| + | |
| - | | + | |
| | <br> | | <br> |
| - | ==LB Agar== | + | <table valign="center" border="0" width="100%"> |
| - | * Add:
| + | <tr><td width="33%" align="center"> |
| - | ** 10 g/L NaCl
| + | <html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Material_Methods/Protocols"> |
| - | ** 10 g/L Bacto-Tryptone
| + | <img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_Protocolli.jpg" width="200px" height="200px"/></a> |
| - | ** 0.5 g/L Bacto-Yeast Extract
| + | </html></td> |
| - | ** 15 g/L Bacto-Agar
| + | <td width="33%" align="center"><html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Material_Methods/Instruments"> |
| - | ** ddH2O
| + | <img src="https://static.igem.org/mediawiki/2010/c/c3/UNIPV_Pavia_strumentazione.jpg" width="200px" height="200"/></a> |
| - | to a sterile 1L flask
| + | </html></td> |
| - | * autoclave
| + | <td width="33%" align="center"><html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Material_Methods/Measurements/Tecan"> |
| - | * (add antibiotic when it reaches ~45°C, shake gently to avoid bubbles)
| + | <img src="https://static.igem.org/mediawiki/2010/3/38/UNIPV_Pavia_provette.jpg" width="200px" height="200px"/></a> |
| - | * pour into Petri plates
| + | </html></td> |
| - | * let them polymerize for ~2-3h
| + | </tr> |
| - | * invert plates and wrap them with aluminium foil and store at +4°C
| + | <tr><td width="33%" align="center"><font size="3">[[Team:UNIPV-Pavia/Material_Methods/Protocols|PROTOCOLS]]</font></td> |
| - | | + | <td width="33%" align="center"><font size="3">[[Team:UNIPV-Pavia/Material_Methods/Instruments|INSTRUMENTS]]</font></td> |
| - | <br> | + | <td width="33%" align="center"><font size="3">[[Team:UNIPV-Pavia/Material_Methods/Measurements/Tecan|TECAN MEASUREMENTS]]</font></td> |
| - | ==SOB== | + | </tr> |
| - | * Add:
| + | </table> |
| - | ** 5 g/L Bacto-Yeast Extract
| + | </td> |
| - | ** 20 g/L Bacto-Tryptone
| + | </tr> |
| - | ** 10mM NaCl
| + | </table> |
| - | ** 2.5mM KCl
| + | |
| - | ** 10mM MgSO4
| + | |
| - | ** 10mM MgCl2
| + | |
| - | to a sterile pyrex bottle
| + | |
| - | * (optional: check that pH is ~6.8, otherwise adjust with NaOH)
| + | |
| - | * autoclave
| + | |
| - | * (add antibiotic when it reaches ~45°C)
| + | |
| - | * store at +4°C
| + | |
| - | | + | |
| - | <br> | + | |
| - | ==SOC== | + | |
| - | * SOB+20mM (3.6 g/L) of glucose (add filter-sterilized (0.2um) glucose to autoclaved SOB).
| + | |
| - | | + | |
| - | <br> | + | |
| - | ==M9 supplemented with glycerol (M9gly)== | + | |
| - | | + | |
| - | For 1L of medium, add:
| + | |
| - | * 716 ml of autoclaved (and cooled to Tamb) ddH2O
| + | |
| - | * 200 ul of autoclaved or filtered (0.2um) CaCl2 0.5 M
| + | |
| - | * 200 ml of autoclaved M9 salts 5x (dissolve 56.4 g in 1 liter ddH2O = 5x stock)
| + | |
| - | * 34 ml of filtered (0.2um) thiamine hydrochloride MW=337.27g/mol (340 mg in 34 ml)
| + | |
| - | * 20 ml of autoclaved MgSO4 0.1 M
| + | |
| - | * 20 ml of 10% autoclaved casamino acids (dissolve 50 g in 500 ml = 10% stock)
| + | |
| - | * 10 ml of autoclaved 40% glycerol as carbon source
| + | |
| - | * mix all the solutions in sterility (each solution must be completely dissolved!)
| + | |
| - | * (add antibiotic)
| + | |
| - | * store at +4°C, protected from light
| + | |
| - | | + | |
| - | NOTE:
| + | |
| - | * M9 salts 5x
| + | |
| - | * 10% casamino acids
| + | |
| - | can be stored at +4°C
| + | |
| - | * MgSO4 0.1 M
| + | |
| - | * CaCl2 0.5 M
| + | |
| - | * glycerol 40%
| + | |
| - | can be stored at room temperature or +4°C
| + | |
| - | * thiamine hydrochloride (LIGHT SENSITIVE) is one-shot and must be prepared each time
| + | |
| - | | + | |
| - | <br> | + | |
| - | | + | |
| - | ==Antibiotics== | + | |
| - | | + | |
| - | Stocks at -20°C freezer:
| + | |
| - | * Ampicillin 100 mg/ml (in water)
| + | |
| - | * Kanamycin 50 mg/ml (in water)
| + | |
| - | * Chloramphenicol 34 mg/ml (in 100% ethanol)
| + | |
| - | These stocks are 1000x for high copy number plasmids.
| + | |
| - | For low copy number plasmids, you should use these final concentrations in media:
| + | |
| - | * Ampicillin 50 ug/ml
| + | |
| - | * Kanamycin 20 ug/ml
| + | |
| - | * Chloramphenicol 12.5 ug/ml
| + | |
| - | | + | |
| | <br><br> | | <br><br> |
| - |
| |
| - | =E. coli transformation=
| |
| - |
| |
| - | ==Transforming home-made competent cells==
| |
| - |
| |
| - | * heat ligation at 65°C to inactivate T4 ligase
| |
| - | * thaw in ice a vial of TOP10 competent cells stored at -80°C
| |
| - | * incubate a selective LB agar plate at 37°C
| |
| - | * pipet 800ul of LB (without antibiotic) in a 15ml falcon tube and incubate it at 37°C
| |
| - | * heat the water bath at 42°C
| |
| - |
| |
| - | * add 1 ul (~3ng of DNA vector) of ligation to 100ul of thawed TOP10
| |
| - | * add parafilm and incubate in ice for 30 min
| |
| - | * heat shock at 42°C for 1 min
| |
| - | * incubate in ice for 2 min
| |
| - | * transfer transformed bacteria to 800ul of pre-warmed LB
| |
| - | * incubate at 37°C, 220 rpm for 1 h
| |
| - | * centrifuge at 1200 rpm, 25°C for 10 min
| |
| - | * take 650ul of supernatant and resuspend the pellet in the remaining LB (~150ul)
| |
| - | * plate the entire culture and incubate the plate at 37°C overnight
| |
| - |
| |
| - |
| |
| - | Variants:
| |
| - | * if you transform a miniprep, add less than 3 ng in order to have single colonies
| |
| - | * if you use another home-made competent strain, the protocol is the same but you should consider the transformation efficiency to add a proper amount of DNA
| |
| - | * if you use commercial Invitrogen TOP10 the protocol changes and it is reported below.
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | Transforming commercial competent cells
| |
| - | (according to manufacturer’s protocol)
| |
| - |
| |
| - | * heat ligation at 65°C to inactivate T4 ligase
| |
| - | * thaw in ice a vial of TOP10 competent cells stored at -80°C (one vial contains 50ul of cells)
| |
| - | * incubate a selective LB agar plate at 37°C
| |
| - | * heat the water bath at 42°C
| |
| - |
| |
| - | * dilute the ligation 1:50 (or 1:100) in ddH2O, in order to have less than 100pg/ul
| |
| - | * add 1 ul of ligation (or less than 100pg of miniprepped DNA) to 25 or 50ul of thawed TOP10
| |
| - | * add parafilm and incubate in ice for 10 min
| |
| - | * heat shock at 42°C for 1 min
| |
| - | * incubate in ice for 2 min
| |
| - | * add 250ul of SOC medium
| |
| - | * incubate at 37°C, 220 rpm for 1 h
| |
| - | * plate 150ul of the culture and incubate the plate at 37°C overnight
| |
| - | * the remaining 150ul can be stored at +4°C
| |
| - |
| |
| - |
| |
| - |
| |
| - | </td></tr>
| |
| - | </table>
| |
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