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

Line 156: Line 156:
  
 
<ol>
 
<ol>
−
<li>Centrifuge with 13000rpm at 24&deg;C for 2min</li>
+
<li>Centrifuge with 13000rpm at 24&deg;C for 2 min</li>
 
<li>Remove the supernatant</li>
 
<li>Remove the supernatant</li>
 
<li>Add 50 mL of SDS-Buffer</li>
 
<li>Add 50 mL of SDS-Buffer</li>

Revision as of 13:00, 18 October 2016

Team:HokkaidoU Japan - 2016.igem.org

 

Team:HokkaidoU Japan

\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\


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 Table. 2 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 100 µL out of each samples and made the following operation.
  1. Centrifuge with 13000rpm at 24°C for 2 min
  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 µL 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.