"
Team:HIT-Harbin/Notebook
From 2011.igem.org
| Line 52: | Line 52: | ||
*<p>An overnight culture grown at 37°C was inoculated in 2.5% glycocoll SMRS as 2% (37°C) and incubated until the optical density at 600 nm (OD600) was 0.5. Cells were harvested by centrifugation (4,000 ×g for 10 min at 4°C) and washed twice with 1 volume of ice-cold electroporation buffe (1 mM KH<sub>2</sub>PO<sub>4</sub>, 0.5 M saccharose , 1 mM MgCl<sub>2</sub>). Finally, pelleted cell were resuspended in 2ml ice-cold electroporation buffer, divided into aliquots and frozen in an ethanol-dry ice bath. Electrocompetent L.bulgaricus cells were stored at -80°C.</p> | *<p>An overnight culture grown at 37°C was inoculated in 2.5% glycocoll SMRS as 2% (37°C) and incubated until the optical density at 600 nm (OD600) was 0.5. Cells were harvested by centrifugation (4,000 ×g for 10 min at 4°C) and washed twice with 1 volume of ice-cold electroporation buffe (1 mM KH<sub>2</sub>PO<sub>4</sub>, 0.5 M saccharose , 1 mM MgCl<sub>2</sub>). Finally, pelleted cell were resuspended in 2ml ice-cold electroporation buffer, divided into aliquots and frozen in an ethanol-dry ice bath. Electrocompetent L.bulgaricus cells were stored at -80°C.</p> | ||
*<p>Electrocompetent cells were thawed on ice, and an 50μl cell suspension was mixed with 1μg of recombinant plasmid DNA. After incubation for 30min on ice, the cells were transferred to an electroporation cuvette with a 0.2cm gap between the elec- trodes. A single pulse of 1.2kV lasting 2.5ms was delivered. The electroporated cells were immediately resuspended in 950μl SMRSMC and incubated for 2h at 37°C, before they were spread on MRS plates containing 2μg/ml erythromycin. Transformants were picked following 24 to 48h of incubation at 37°C.</p> | *<p>Electrocompetent cells were thawed on ice, and an 50μl cell suspension was mixed with 1μg of recombinant plasmid DNA. After incubation for 30min on ice, the cells were transferred to an electroporation cuvette with a 0.2cm gap between the elec- trodes. A single pulse of 1.2kV lasting 2.5ms was delivered. The electroporated cells were immediately resuspended in 950μl SMRSMC and incubated for 2h at 37°C, before they were spread on MRS plates containing 2μg/ml erythromycin. Transformants were picked following 24 to 48h of incubation at 37°C.</p> | ||
| - | |||
| - | |||
==='''The competent cell of Streptococcus thermophilus'''=== | ==='''The competent cell of Streptococcus thermophilus'''=== | ||
| - | + | ||
</div> | </div> | ||
| + | |||
=='''PCR Reaction'''== | =='''PCR Reaction'''== | ||
<div class="cadre"> | <div class="cadre"> | ||
Revision as of 03:12, 6 October 2011
Contents |
Weekly Diary
Protocal
The Composition Of Media
LB Medium
Yeast extact 5g
Peptone 10g
NaCl 10g
Agar 1-2%
Distilled water 1000ml pH 7.0
Boil the mixture in autoclave at 121℃ for 30min
Range of applicationb: Escherichia.coli
MRS Medium
Peptone 10g
Beef extract 10g
Yeast extact 5g
K2HPO4 2g
Tween 80 1ml
Diamine citrate 2g
Na-acetate 5g
MgSO4.7H2O 0.5g
MnSO4.H2O 0.25g
Glucose 20g
Agar 1-2%
Distilled water 1000ml Adjust pH to 6.4-6.8
Boil the mixture in autoclave at 115℃ for 20min
Range of application: Lactobacillus bulgaricus
2.5% glycocoll SMRS is MRS supplemented with 2.5% glycocoll 0.5mol/L saccharose, 20mmol/L MgCl2,20mmol/L CaCl2
SMRSMC is MRS supplemented with 0.5mol/L saccharose, 20mmol/L MgCl2,20mmol/L CaCl2
Hogg-Jago glucose broth(HJG) medium
Tryptone 30g
Yeast extract 10g
Beef extract 2g
KH2PO4 5g
Glucose 5g
Agar 1.5%
Distilled water 1000ml
Boil the mixture in autoclave at 115℃ for 20min
Range of application: Streptococcus thermophilus
HJGL is Hogg-Jago glucose broth supplemented with 0.5% lactose, whereas HJGLS is HJGL supplemented with 0.4M D-sorbitol
Agar plates were prepared by adding 1.5% (wt/vol) agar to the media
Competent Cell and Electroporation
The competent cell of Lactobacillus bulgaricus
An overnight culture grown at 37°C was inoculated in 2.5% glycocoll SMRS as 2% (37°C) and incubated until the optical density at 600 nm (OD600) was 0.5. Cells were harvested by centrifugation (4,000 ×g for 10 min at 4°C) and washed twice with 1 volume of ice-cold electroporation buffe (1 mM KH2PO4, 0.5 M saccharose , 1 mM MgCl2). Finally, pelleted cell were resuspended in 2ml ice-cold electroporation buffer, divided into aliquots and frozen in an ethanol-dry ice bath. Electrocompetent L.bulgaricus cells were stored at -80°C.
Electrocompetent cells were thawed on ice, and an 50μl cell suspension was mixed with 1μg of recombinant plasmid DNA. After incubation for 30min on ice, the cells were transferred to an electroporation cuvette with a 0.2cm gap between the elec- trodes. A single pulse of 1.2kV lasting 2.5ms was delivered. The electroporated cells were immediately resuspended in 950μl SMRSMC and incubated for 2h at 37°C, before they were spread on MRS plates containing 2μg/ml erythromycin. Transformants were picked following 24 to 48h of incubation at 37°C.
The competent cell of Streptococcus thermophilus
PCR Reaction
Agarose Gel Electrophoresis
Gel Purification of DNA (Solarbio QIAquick Gel Extraction Kit)
