Team:Kyoto/GSDD/Notebook/0921-0927

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Construction.
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Construction.PCR.
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Construction.PCR.MPR.
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Construction.PCR.MPR.
0921-0927
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1005-1011
Construction.PCR.MPR.
1012-1018
Construction.MPR.Observation.

0921-0927 : Construction.PCR.MPR.


Monday, 21 September

To Do

  1. Colony PCR
    • Insert check
  1. Miniprep
    • Restriction enzyme digestion
    • Gel extraction and PCR purification
    • Ligation
    • Transformation

Results

PCR

Kyoto 0921 1.png

  • ①and ②and ③ are inserted properly.
Miniprep
sampleconc./(ng/ul)
4(1)70.3
4(2)54.8
4(3)a84.7
1-O-265.1

Kyoto 0921 2.png

Gel extraction
4(1)a E-S,1-0-2 X-P
PCR purification
4(2) E-X



Tuesday, 22 September

To Do

  1. Miniprep 3(1)a1 4(4)a1 11①
    • Restriction enzyme digestion
    • Gel extraction and PCR purification
    • Ligation
  1. MPR


Results

PCR

Kyoto 0922 1.png Gel extraction and PCR purification Sample 4(4)E-X, 3(1)E-S


Gel extraction

Kyoto 0922 2.png

sampleconc./(ng/ul)
MPR124.8
MPR281.0
MPR360.1
MPR431.2



Wednesday, 23 September

To Do

  1. Phosphorylation
  2. Miniprep
    • Restriction enzyme digestion
    • BAP Gel extraction and PCR purification
    • Ligation

Results

electrophoresis

Kyoto 0923 1.png 11-1 can be cut by stuI. 4(5) can not be inserted in Byp 561 because 4(5) has PstI site.

Dephosphorylation

Kyoto 0923 2.png

  • Gel extraction.
sampleconc./(ng/ul)
4(4)X-P22.3
11-1a BAP25.1
  • PCR purification
sampleconc./(ng/ul)
4(3)S-P134.3
MPR131.1
MPR252.2



Thursday, 24 September

To Do

  1. Restriction enzyme digestion (Stu1)
    • Transformation
  1. Transformation
  2. Miniprep
    • Restriction enzyme digestion
    • Ligation
    • Transformation
  1. Ligation
    • transformation

Results

Miniprep
sampleconc./(ng/ul)
3(2)36.0
4(5)a39.6
4(5)b34.7
4(5)c37.4



Friday, 25 September

To Do

  1. Culture 1M 2M(1) 11 (1)
    • Restriction enzyme digestion
    • Insert check
  1. Electorophoresis 4(5)a,b,c
  2. Colony PCR


Results

Electrophoresis

Kyoto 0925.png 1M contains product of MPR with 200bp. 2M ,at first, contains product of MPR with 1kbp. With transformed, it changed to with 600bp.