Difference between revisions of "Team:HokkaidoU Japan/Proof"

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   The following Table2 is about preparing the SDS-PAGE.  
 
   The following Table2 is about preparing the SDS-PAGE.  
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　　After we finished cultivating ?samples, we took 100μl out of each samples and made the following operation.
 
　　After we finished cultivating ?samples, we took 100μl out of each samples and made the following operation.
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<br>
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<ol>
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<li>Centrifuge with 13000rpm at 24&deg;C for 2min</li>
 +
<li>Remove the supernatant</li>
 +
<li>Add 50 mL of SDS-Buffer</li>
 +
<li>Shake for 1 min</li>
 +
<li>Keep at 100&deg;C for 5 min</li>
 +
<li>Put on the ice</li>
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<li>Apply 10 &micro; to SDS</li>
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<li>Run electrophoresis for 1.5 h</li>
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<li>Wash out with MiliQ</li>
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<li>Shake at 24&deg;C with 34 rpm for 1 h</li>
 +
<li>Dye with QuickCBB</li>
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</ol>
  
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1. Centrifuge with 13000rpm at 24℃ for 2min
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<br>
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2. Remove the supernatant
+
−
3. Add 50ml of SDS-Buffer
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−
4. Shake for 1min
+
−
5. Keep at 100℃ for 5min
+
−
6. Put on the ice
+
−
7. Apply 10μl to SDS 
+
−
8. Run electrophoresis for 1.5h
+
−
9. Wash out with MiliQ
+
−
10. Shake at 24℃ with 34rpm for 1h
+
−
11. Dye with QuickCBB
+
  
 
The fig. 1 shows the result of SDS-PAGE.
 
The fig. 1 shows the result of SDS-PAGE.

Revision as of 12:53, 18 October 2016

Team:HokkaidoU Japan - 2016.igem.org

 

Team:HokkaidoU Japan

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Proof of concept

We conducted SDS-PAGE with (BBa_K2015012+BBa_K2015008/ BBa_K2015012+BBa_K2015009) on （pSB1C3）of E.coli（DH5α）. At first, we made the Gel for SDS-PAGE, with the following the Table1. What was the most important was to add TEMED ffinally because

Table. 1. Gel assay
Materials Volume
30％ Acrilmid 5 mL
Tris-Buffer 2.5 mL
SDS 100 µL
APS 100 µL
TEMED 5 µL
DW 2.295 ｍL
Total 10 ｍL


The following Table2 is about preparing the SDS-PAGE.

Table. 2. Experimental condition
Temperature IPTG Concentration (M) Volume (µL) Time to culture
37°C - - - 24 h
37°C + 0.4 6 24 h
37°C + 2 30 24 h
25°C - - - 16 h
25°C + 0.4 6 16 h
25°C + 2 30 16 h

　　After we finished cultivating ?samples, we took 100μl out of each samples and made the following operation.
  1. Centrifuge with 13000rpm at 24°C for 2min
  2. Remove the supernatant
  3. Add 50 mL of SDS-Buffer
  4. Shake for 1 min
  5. Keep at 100°C for 5 min
  6. Put on the ice
  7. Apply 10 µ to SDS
  8. Run electrophoresis for 1.5 h
  9. Wash out with MiliQ
  10. Shake at 24°C with 34 rpm for 1 h
  11. Dye with QuickCBB

The fig. 1 shows the result of SDS-PAGE.