Team:UNIPV-Pavia/Material Methods

From 2010.igem.org

(Difference between revisions)
 
(25 intermediate revisions not shown)
Line 1: Line 1:
-
<!-- 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
 
-
 
-
<br><br>
 
-
 
-
=E. coli competent cells preparation=
 
-
H. Inoue et al. (1990), High efficiency transformation of Escherichia coli with plasmids, Gene 96 23-28.
 
-
 
-
; DAY1
 
-
: inoculum 5-8 ul from -80°C stock in 5 ml of LB (37°C, 220 rpm ON);
 
-
;DAY2
 
-
: dilution 1:1000 in SOB (flask, 18-25°C, 220 rpm ON);
 
-
; DAY3
 
-
: pre-chill centrifuge at 4°C;
 
-
: prepare TB (prepare 50 ml every 125 ml of SOB):
 
-
::* 15mM CaCl2
 
-
::* 250mM KCl
 
-
::* 10mM (3 g/L) Pipes
 
-
::* adjust pH at 6.7 with KOH
 
-
::* 55mM (8.9 g/L) MnCl2
 
-
::* filter (0.2 um) the solution and chill) in 50 ml
 
-
: put the flask in ice when the culture reaches OD600=~0.05 (1mm pathlength – NanoDrop);
 
-
: aliquot in pre-chilled 50 ml falcon tubes;
 
-
: 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);
 
-
 
-
<br><br>
 
-
 
-
=E. coli strains (all in -80°C freezer)=
 
-
 
-
 
-
==TOP10==
 
-
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
 
-
 
-
 
-
==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
 
-
 
-
 
-
==BW20767==
 
-
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
 
-
NOTE: they have
 
-
* a fully working lac operon (already tested on IPTG/X-Gal plates)
 
-
* Kan and Tet resistance (not tested)
 
-
 
-
 
-
==XL1-Blue==
 
-
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
 
-
 
-
 
-
 
-
 
-
 
-
 
-
==DB3.1==
 
-
F- gyrA462 endA1 glnV44 Δ(sr1-recA) mcrB mrr hsdS20(rB-, mB-) ara14 galK2 lacY1 proA2 rpsL20(Smr) xyl5 Δleu mtl1
 
-
* source: Francesca Ceroni
 
-
* 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
 
-
 
-
 
-
==STBL3==
 
-
F- glnV44 recA13 mcrB mrr hsdS20(rB-, mB-) ara-14 galK2 lacY1 proA2 rpsL20 xyl-5 leu mtl-1
 
-
* source: Invitrogen
 
-
* competent cells never prepared
 
-
* used for in vivo amplification of DNA with direct repeats
 
-
NOTE: they cannot be used for blue/white screening
 
-
 
-
 
-
==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
 
-
 
-
 
-
 
-
 
-
</td></tr>
 
-
</table>
 

Latest revision as of 15:00, 22 October 2010
Home
The Project
Materials & Methods
Parts
The Team
Notebook
Biological Safety
Pavia
Gallery
Sponsors


Material & Methods


PROTOCOLS INSTRUMENTS TECAN MEASUREMENTS



Retrieved from "http://2010.igem.org/Team:UNIPV-Pavia/Material_Methods"