Team:Chiba/Assay/uptake

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<h2>uptake実験方法</h2>
<h2>uptake実験方法</h2>
<p>
<p>
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1.ホニャララ<br>
+
1.E.coli strain BL21 were transformed with the plasmids shown in Fig. 1 and Fig.2.<br>
-
2.かにやら<br>
+
2.All transformants were inoculated into culture(100 mL TB medium) and was shaken for 12h (at 37℃).<br>
-
3.なんやかんや<br>
+
3.We centrifuged samples with 3,750 rpm, at 4℃ for 25 minutes and resuspended with 5 mL LB medium.<br>
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4.なんやかんや<br>
+
4.We added L-arabinose into each sample (final conc. 0.2%). Then cultured for another 9 hours (at 37℃).<br>
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5.<br>
+
5.We diluted samples by LB medium and got 500 uL of the diluted cell suspension. Approximately, 2×10^9, 10^9, 5×10^8 cells were contained in it.<br>
 +
6.We added 5 uL from 10 mM iron citrate(Ⅲ) and 5uL from 10 mM iron ascorbate(Ⅱ) to the sample, and we shake the sample for 1h (at 37℃) to charge iron inside E.coli.<br>
 +
7.We centrifuged the sample with 19,600 rpm, at 37℃ for 1 minutes and got supernatant(60 mL).<br>
 +
8.We added color reagent, Tris buffer solution, Reducing Agent to the sample and measured absorbance (Abs756) to determine the density of iron.<br>
絵とかあるといいよね!!
絵とかあるといいよね!!

Revision as of 04:59, 27 September 2013

iGEM-2013 Chiba

iGEM-2013 Chiba

uptake実験方法

1.E.coli strain BL21 were transformed with the plasmids shown in Fig. 1 and Fig.2.
2.All transformants were inoculated into culture(100 mL TB medium) and was shaken for 12h (at 37℃).
3.We centrifuged samples with 3,750 rpm, at 4℃ for 25 minutes and resuspended with 5 mL LB medium.
4.We added L-arabinose into each sample (final conc. 0.2%). Then cultured for another 9 hours (at 37℃).
5.We diluted samples by LB medium and got 500 uL of the diluted cell suspension. Approximately, 2×10^9, 10^9, 5×10^8 cells were contained in it.
6.We added 5 uL from 10 mM iron citrate(Ⅲ) and 5uL from 10 mM iron ascorbate(Ⅱ) to the sample, and we shake the sample for 1h (at 37℃) to charge iron inside E.coli.
7.We centrifuged the sample with 19,600 rpm, at 37℃ for 1 minutes and got supernatant(60 mL).
8.We added color reagent, Tris buffer solution, Reducing Agent to the sample and measured absorbance (Abs756) to determine the density of iron.
絵とかあるといいよね!!