https://gmhwiki.alexandersci.com/index.php?action=history&feed=atom&title=Streptococcus_transformation_protocolStreptococcus transformation protocol - Revision history2026-11-14T21:09:57ZRevision history for this page on the wikiMediaWiki 1.46.2https://gmhwiki.alexandersci.com/index.php?title=Streptococcus_transformation_protocol&diff=1147&oldid=prevAhetta: Streptococcus transformation protocol2026-10-27T23:10:32Z<p>Streptococcus transformation protocol</p>
<p><b>New page</b></p><div>'''Culture media:'''<br />
<br />
'''1. For overnight culture (SGM17):'''<br />
<br />
Beef extract ……………….. 5.00 g/l<br />
<br />
Polypeptone ……………… 5.00 g/l <br />
<br />
Phytone-peptone …………… 5.00g/l<br />
<br />
Yeast extract ……………….. 2.50 g/l <br />
<br />
Disodium glycerol phosphate 19.00 g/l<br />
<br />
Ascorbic acid ………………...0.50 g/l<br />
<br />
Glucose ………………………5.00 g/l<br />
<br />
MgSO4.7H2O ………………. 0.25 g/l<br />
<br />
+<br />
<br />
0.5 M Sucrose (171.15 g/l)<br />
<br />
- Filter-sterilize <br />
<br />
'''2. For growing the cells to make electrocompetent cells:'''<br />
<br />
SGM17 (above) + '''0.5% glycine''' <br />
<br />
'''3. For recovery after electroporation:'''<br />
<br />
SGM17 containing '''20 mM MgCl2''' and '''2 mM CaCl2''' <br />
<br />
'''4. For plating:'''<br />
<br />
'''SR medium contains (g/l):''' (Supplement with erythromycin (5 and 10 mg/ml)<br />
<br />
- Tryptone 10 <br />
<br />
- Yeast extract 5.0 <br />
<br />
- Sucrose 200 <br />
<br />
- glucose 10 <br />
<br />
- Gelatin, 25 <br />
<br />
- agar 15 <br />
<br />
- MgCl 2.5 mM <br />
<br />
- CaCl<sub>2</sub> 2.5 mM <br />
<br />
<nowiki>*</nowiki>Pour plates in the anaerobic chamber. <br />
<br />
'''Method:'''<br />
<br />
'''Day1'''. Grow Streptococcus in 10 ml of GM17+Sucrose culture medium overnight aerobically. <br />
<br />
'''Day2'''. '''Preparation of competent cells'''<br />
<br />
- Inoculate prewarmed 250 ml of GM17+Sucrose +'''0.5% glycine''' culture medium with 5 ml of the overnight culture.<br />
<br />
- Grow aerobically at 37 °C to an OD600 of 0.2 (approximately 5 to 7 h).<br />
<br />
- Place the culture on ice for 30 minutes and centrifuge at 5000 g, 4 °C, for 15 min.<br />
<br />
- Wash the cell pellets three times in 25 ml of ice-cold 0.5 M sucrose/10% glycerol, and centrifuge as before''.''<br />
<br />
- Resuspend the cell pellet in 0.5 ml of 0.5 M sucrose/10% glycerol and either use immediately for electroporation or store at -80 °C. <br />
<br />
'''Electroporation:'''<br />
<br />
- Mix 100 ml of the cell suspension was mixed with up to 5 ml of DNA and place in ice-cold 0.2-cm electroporation cuvette (Bio-Rad Laboratories, Richmond, CA). <br />
<br />
- Electroporate at 2.5 kV, 200 W, 25 mF using the Bio-Rad Gene Pulser. <br />
<br />
- Immediately after electroporation, add 0.9 ml of SGM17 containing '''20 mM MgCl2 and 2 mM CaCl2''' to the cell suspension.<br />
<br />
- Transfer to a tube and incubate the mixture at 37 °C for 1 h.<br />
<br />
- Dilutions of the cells were plated on SR and SDM agar plates supplemented with antibiotic (5 mg/ml erythromycin).<br />
<br />
- Incubate in the anaerobic chamber and incubate at 37 °C for 1-2 days.</div>Ahetta