Team:UNIPV-Pavia/Material Methods

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      <td colspan="2"> {{UNIPV-Pavia/header}} </td> 
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    <td colspan="2">{{UNIPV-Pavia/header}}</td>
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<tr><td align="left" valign="top" width="20%">{{UNIPV-Pavia/menu}}</td><td>
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<table valign="center" border="0" width="100%">
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<tr><td align="left" valign="top" width="20%">{{UNIPV-Pavia/menu}}</td>
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Material_Methods/Protocols">
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<img src="https://static.igem.org/mediawiki/2010/thumb/7/7b/UNIPV_Pavia_Calendario.jpg/200px-UNIPV_Pavia_Calendario.jpg"/></a>
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<!-- Contenuti -->
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</html></td>
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    <td width="33%" align="center"><html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Material_Methods/Instruments">
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<td valign="top" >
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<img src="https://static.igem.org/mediawiki/2010/0/0f/UNIPV_Pavia_Topics.jpg" width="300px"/></a>
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<html><a name="indice"/></html>
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</html></td>
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=Media & Antibiotics=
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    <td width="33%" align="center"><html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Material_Methods/Measurements">
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<img src="https://static.igem.org/mediawiki/2010/thumb/8/88/UNIPV_Pavia_Congelatore.jpg/200px-UNIPV_Pavia_Congelatore.jpg"/></a>
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==LB==
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</html></td>
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* Add:
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</tr>
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** 10 g/L NaCl
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<tr><td width="33%" align="center"><font size="3">[[Team:UNIPV-Pavia/Material_Methods/Protocols|PROTOCOLS]]</font></td>
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** 10 g/L Bacto-Tryptone
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    <td width="33%" align="center"><font size="3">[[Team:UNIPV-Pavia/Material_Methods/Instruments|INSTRUMENTS]]</font></td>
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** 0.5 g/L Bacto-Yeast Extract
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    <td width="33%" align="center"><font size="3">[[Team:UNIPV-Pavia/Material_Methods/Measurements|MEASUREMENTS]]</font></td>
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** ddH2O
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</tr>
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to a sterile pyrex bottle
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</table>
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* autoclave
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* (add antibiotic when it reaches ~45°C)
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* store at +4°C
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<div align="right"><small>[[#indice|^top]]</small></div>
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-
<br>
+
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+
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==LB Agar==
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* Add:
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** 10 g/L NaCl
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** 10 g/L Bacto-Tryptone
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** 0.5 g/L Bacto-Yeast Extract
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** 15 g/L Bacto-Agar
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** ddH2O
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to a sterile 1L flask
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* autoclave
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* (add antibiotic when it reaches ~45°C, shake gently to avoid bubbles)
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* pour into Petri plates
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* let them polymerize for ~2-3h
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* invert plates and wrap them with aluminium foil and store at +4°C
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<div align="right"><small>[[#indice|^top]]</small></div>
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<br>
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+
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==SOB==
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* Add:
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** 5 g/L Bacto-Yeast Extract
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** 20 g/L Bacto-Tryptone
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** 10mM NaCl
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** 2.5mM KCl
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** 10mM MgSO4
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** 10mM MgCl2
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to a sterile pyrex bottle
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* (optional: check that pH is ~6.8, otherwise adjust with NaOH)
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* autoclave
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* (add antibiotic when it reaches ~45°C)
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* store at +4°C
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<div align="right"><small>[[#indice|^top]]</small></div>
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<br>
+
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==SOC==
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* SOB+20mM (3.6 g/L) of glucose (add filter-sterilized (0.2um) glucose to autoclaved SOB).
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<div align="right"><small>[[#indice|^top]]</small></div>
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<br>
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==M9 supplemented with glycerol (M9gly)==
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For 1L of medium, add:
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* 716 ml of autoclaved (and cooled to Tamb) ddH2O
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* 200 ul of autoclaved or filtered (0.2um) CaCl2 0.5 M
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* 200 ml of autoclaved M9 salts 5x (dissolve 56.4 g in 1 liter ddH2O = 5x stock)
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* 34 ml of filtered (0.2um) thiamine hydrochloride MW=337.27g/mol (340 mg in 34 ml)
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* 20 ml of autoclaved MgSO4 0.1 M
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* 20 ml of 10% autoclaved casamino acids (dissolve 50 g in 500 ml = 10% stock)
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* 10 ml of autoclaved 40% glycerol as carbon source
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* mix all the solutions in sterility (each solution must be completely dissolved!)
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* (add antibiotic)
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* store at +4°C, protected from light
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NOTE:
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* M9 salts 5x
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* 10% casamino acids
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can be stored at +4°C
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* MgSO4 0.1 M
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* CaCl2 0.5 M
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* glycerol 40%
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can be stored at room temperature or +4°C
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* thiamine hydrochloride (LIGHT SENSITIVE) is one-shot and must be prepared each time
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<div align="right"><small>[[#indice|^top]]</small></div>
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+
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<br>
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==Antibiotics==
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Stocks at -20°C freezer:
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* Ampicillin 100 mg/ml (in water)
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* Kanamycin 50 mg/ml (in water)
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* Chloramphenicol 34 mg/ml (in 100% ethanol)
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These stocks are 1000x for high copy number plasmids.
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For low copy number plasmids, you should use these final concentrations in media:
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* Ampicillin 50 ug/ml
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* Kanamycin 20 ug/ml
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* Chloramphenicol 12.5 ug/ml
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<div align="right"><small>[[#indice|^top]]</small></div>
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<br><br>
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=E. coli transformation=
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+
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==Transforming home-made competent cells==
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+
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* heat ligation at 65°C to inactivate T4 ligase
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* thaw in ice a vial of TOP10 competent cells stored at -80°C
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* incubate a selective LB agar plate at 37°C
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* pipet 800ul of LB (without antibiotic) in a 15ml falcon tube and incubate it at 37°C
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* heat the water bath at 42°C
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+
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* add 1 ul (~3ng of DNA vector) of ligation to 100ul of thawed TOP10
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* add parafilm and incubate in ice for 30 min
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* heat shock at 42°C for 1 min
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* incubate in ice for 2 min
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* transfer transformed bacteria to 800ul of pre-warmed LB
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* incubate at 37°C, 220 rpm for 1 h
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* centrifuge at 1200 rpm, 25°C for 10 min
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* take 650ul of supernatant and resuspend the pellet in the remaining LB (~150ul)
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* plate the entire culture and incubate the plate at 37°C overnight
+
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+
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+
-
Variants:
+
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* 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.
+
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<div align="right"><small>[[#indice|^top]]</small></div>
+
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+
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<br>
+
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+
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==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
+
-
 
+
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* 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
+
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* add parafilm and incubate in ice for 10 min
+
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* heat shock at 42°C for 1 min
+
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* incubate in ice for 2 min
+
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* add 250ul of SOC medium
+
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* incubate at 37°C, 220 rpm for 1 h
+
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* plate 150ul of the culture and incubate the plate at 37°C overnight
+
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* the remaining 150ul can be stored at +4°C
+
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<div align="right"><small>[[#indice|^top]]</small></div>
+
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+
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<br><br>
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+
-
=E. coli competent cells preparation=
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-
H. Inoue et al. (1990), High efficiency transformation of Escherichia coli with plasmids, Gene 96 23-28.
+
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+
-
; DAY1
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-
: inoculum 5-8 ul from -80°C stock in 5 ml of LB (37°C, 220 rpm ON);
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-
;DAY2
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: dilution 1:1000 in SOB (flask, 18-25°C, 220 rpm ON);
+
-
; DAY3
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: pre-chill centrifuge at 4°C;
+
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: prepare TB (prepare 50 ml every 125 ml of SOB):
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::* 15mM CaCl2
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::* 250mM KCl
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::* 10mM (3 g/L) Pipes
+
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::* adjust pH at 6.7 with KOH
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::* 55mM (8.9 g/L) MnCl2
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::* filter (0.2 um) the solution and chill) in 50 ml
+
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: put the flask in ice when the culture reaches OD600=~0.05 (1mm pathlength – NanoDrop);
+
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: aliquot in pre-chilled 50 ml falcon tubes;
+
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: centrifuge at 2500g (4400rpm), 4°C, 10 min;
+
-
: ICE: discard, resuspend in 40 ml of TB each 125 ml SOB, centrifuge as before;
+
-
: ICE: discard, resuspend in 10 ml of TB each 125 ml SOB, add 700ul DMSO;
+
-
: ICE: aliquot 100ul in pre-chilled 0.5ml tubes;
+
-
: put in -80°C freezer;
+
-
ALWAYS TEST THE EFFICIENCY IN [CFU/ug] UNITS
+
-
 
+
-
This protocol has shown to work with:
+
-
* DH5alpha (10^8 with 100ul of cells);
+
-
* TOP10 (5*10^7 with 100ul of cells);
+
-
* BW20767 (10^3 with 100ul of cells);
+
-
* DB3.1 (5*10^4 with 100ul of cells);
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
 
+
-
<br><br>
+
-
 
+
-
=E. coli strains (all in -80°C freezer)=
+
-
 
+
-
 
+
-
==TOP10==
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-
F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-
+
-
* source: Invitrogen
+
-
* competent cells already prepared (5*10^7 CFU/ug with100ul of cells)
+
-
* competent cells from Invitrogen available (10^9 CFU/ug with 50ul of cells)
+
-
* commonly used for cloning and expression in our lab
+
-
* they are equal to DH10B strain, whose genome is available from NCBI
+
-
NOTE: they have
+
-
* lacI wt
+
-
* cI of phi80 prophage (different from cI of lambda phage)
+
-
* Streptomycin resistance
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
 
+
-
==DH5alpha==
+
-
F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–
+
-
* source: Francesca Ceroni
+
-
* competent cells already prepared (10^8 CFU/ug with100ul of cells)
+
-
* commonly used for cloning
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
 
+
-
==BW20767==
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-
F-, RP4-2(Km::Tn7,Tc::Mu-1), leu-163::IS10, ΔuidA3::pir+, recA1, endA1, thi-1, hsdR17, creC510
+
-
* source: Vinoo Selvarajah
+
-
* competent cells already prepared (10^3 CFU/ug with100ul of cells)
+
-
* not used for cloning
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-
NOTE: they have
+
-
* a fully working lac operon (already tested on IPTG/X-Gal plates)
+
-
* Kan and Tet resistance (not tested)
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
 
+
-
==XL1-Blue==
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-
endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK- mK+)
+
-
* source: Francesca Ceroni
+
-
* competent cells never prepared
+
-
* a small stock of competent cells is available
+
-
* used for cloning
+
-
NOTE: they have lacIQ
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
 
+
-
==DB3.1==
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-
F- gyrA462 endA1 glnV44 Δ(sr1-recA) mcrB mrr hsdS20(rB-, mB-) ara14 galK2 lacY1 proA2 rpsL20(Smr) xyl5 Δleu mtl1
+
-
* source: Francesca Ceroni
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-
* competent cells already prepared (5*10^4 CFU/ug with 100ul of cells)
+
-
* used for in vivo amplification of ccdB plasmids
+
-
NOTE: they have a working lacZ, but a deleted lacY, they become slightly blue on IPTG/X-Gal plates
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
 
+
-
==STBL3==
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-
F- glnV44 recA13 mcrB mrr hsdS20(rB-, mB-) ara-14 galK2 lacY1 proA2 rpsL20 xyl-5 leu mtl-1
+
-
* source: Invitrogen
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-
* competent cells never prepared
+
-
* used for in vivo amplification of DNA with direct repeats
+
-
NOTE: they cannot be used for blue/white screening
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
 
+
-
==CW2553 + pJat8==
+
-
Genotype: Khlebnikov A et al. (2000), Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture, Journal of Bacteriology, Vol. 182, No. 24, p.7029-7034.
+
-
* Source: Vinoo Selvarajah
+
-
* pJat8 is Gentamycine resistant
+
-
NOTE:
+
-
* the stock of this strain has been grown without Gen
+
-
* this strain is used for araBAD inducible system
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
<br><br>
+
-
 
+
-
=Long term bacterial glycerol stocks=
+
-
* Mix 750 ul of a culture (preferably in log-phase) with 250 ul of 80% glycerol, in a 1.5ml vial
+
-
* label the vial with name, date and antibiotic resistance
+
-
* leave at -20°C for one day
+
-
* move to -80°C the day after
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
<br><br>
+
-
 
+
-
=Plasmid digestion for BioBrick Standard Assembly=
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-
; To open vectors:
+
-
:* a volume containing 1 ug of purified plasmid
+
-
:* 2 ul (or 2.5 ul if the final volume is 25 ul) of 10X buffer H
+
-
:* 1 ul of first enzyme
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-
:* 1 ul of second enzyme
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-
:* 20 ul (or 25 ul) final volume
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-
:* incubate at 37°C for 3 hours
+
-
; To excide fragments:
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:* A volume containing 1-2.5 ug of purified plasmid
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-
:* 2.5 ul of buffer H
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-
:* 1 ul of first enzyme
+
-
:* 1 ul of second enzyme
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-
:* 25 ul final volume
+
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:* incubate at 37°C for 3 hours
+
-
NOTE: if you are performing a digestion for screening, 1 hour of incubation is sufficient.
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
<br><br>
+
-
 
+
-
=Ethanol precipitation with sodium acetate=
+
-
 
+
-
* Add 1/10 DNA solution volume of sodium acetate 3 M, pH 5.2
+
-
* Add 2.5 DNA solution volume of absolute ethanol
+
-
* Freeze at -80°C for 30 min
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-
* Centrifuge at 13000 rpm, 4°C for 20 min
+
-
* Decant supernatant
+
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* Add 250 µl of 70% ethanol
+
-
* Centrifuge at 13000 rpm, 4°C for 20 min
+
-
* Remove all supernatant with a pipette
+
-
* Air dry pellet until ethanol is totally removed
+
-
* Elute with 5-10 µl of ddH2O
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
+
-
<br><br>
+
-
 
+
-
=Ligation=
+
-
 
+
-
After the purification of two digested DNA fragments:
+
-
* add a volume containing 20-50 ng of vector
+
-
* add a volume containing:
+
-
[[Image:Pv_formula_lig.png]]
+
-
 
+
-
(“6” can be lowered to “2”)
+
-
* heat DNA mix at 65°C for 5 min for DNA denaturation
+
-
* add 1 ul of T4 Ligase buffer (check if ATP is completely dissolved)
+
-
* add 1 ul of T4 Ligase
+
-
* 10-20 µl final volume
+
-
* incubate at 16°C overnight
+
-
* inactivate the T4 Ligase heating at 65°C for 10 min
+
-
* then, ligation can be conserved at 4°C or can be transformed
+
-
 
+
-
NOTE:
+
-
When the purified DNA of the insert also contains its native vector, you can perform the ligation anyway, but its antibiotic resistance must be different from the acceptor vector’s resistance in order to select correct transformants on agar plates.
+
-
When doing this, you should modify the ligation protocol:
+
-
* you should use “2” or “3” instead of “6” to compute the insert mass;
+
-
* when you add the volume containing the insert mass, you must consider that the DNA quantification with NanoDrop refers to insert+NATIVE VECTOR. So, you must add:
+
-
[[Image:Pv_formula_lig_2.jpeg]]
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
<br/><br/>
+
-
 
+
-
=DNA resuspension from iGEM plates=
+
-
 
+
-
*Find the right position of the DNA of interest in iGEM plates
+
-
*Resuspend with 15 ul ddH2O and transfer it in 0,5 ml sterile Eppendorf tubes
+
-
Now you can store them at -20°C or transform in your favorite strain.
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
<br/><br/>
+
-
 
+
-
=PCR=
+
-
*For every DNA sample you want to amplify, put:
+
-
**2 µl buffer
+
-
**0.6 µl MgCl2
+
-
**0.4 µl dNTPs
+
-
**1 µl DNA (or ddH2O for blank sample). If you are performing a colony PCR, pick up the desired colony from a plate with a tip and dip it in the solution.
+
-
**0.2 µl Taq Polymerase
+
-
**250 nM VF2 primer
+
-
**250 nM VR primer
+
-
**A proper amount of ddH2O to have 20 µl of total reaction volume
+
-
*into an eppendorf tube.
+
-
*Put the Eppendorf tube in the thermal cycler and set this program:
+
-
**95°C 10 min
+
-
**CYCLE:
+
-
***95°C 30 sec
+
-
***60°C 1 min
+
-
***72°C 1-3 min
+
-
**for 35 cycles
+
-
**72°C 7 min
+
-
**16°C forever.
+
-
*Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length.
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
<br/><br/>
+
-
 
+
-
=Electrophoresis=
+
-
<table><tr>
+
-
<td valign="top">
+
-
*Prepare agarose gel in 1x TBE buffer
+
-
*Add ethidium bromide (using gloves and face mask for your safety):
+
-
**1,5 µl in the small size agarose gel (70 ml)
+
-
**3 µl in the middle size agarose gel (150 ml)
+
-
**5 µl in the big size agarose gel (250 ml)
+
-
*Cast the gel, insert the well-forming comb and let it polymerize
+
-
*Add the loading buffer (10x Blue Juice, Invitrogen) to each sample
+
-
*Load the samples and 8 µl of marker (when not specified: 1 kb Plus DNA Ladder, Fermentas)
+
-
*Set to 70-100 volts and electrophorese for the required amount of time
+
-
*Use UV-light to look at the bands (using gloves and protective glasses)
+
-
*Take a picture of the gel, if needed (not when bands have to be cut!!!)
+
</td>
</td>
-
<td valign="top">
+
</tr>
-
[[Image:1kb_unipv.jpg|thumb|150px|1kb marker]]
+
-
</td>
+
-
</tr></table>
+
-
 
+
-
<div align="right"><small>[[#indice|^top]]</small></div>
+
-
<br/><br/>
+
-
 
+
-
</td></tr>
+
</table>
</table>

Revision as of 15:19, 23 June 2010
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