Team:Kyoto/GSDD/Notebook/0907-0911

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Construction.
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Construction.
0810-0816
Construction.
0817-0823
Construction.PCR.
0824-0830
Construction.PCR.
0831-0906
Construction.PCR.MPR.
0907-0913
Construction.PCR.MPR.
0914-0920
Construction.PCR.MPR.
0921-0927
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0928-1004
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1005-1011
Construction.PCR.MPR.
1012-1018
Construction.MPR.Observation.

0907-0911 : what to do. what to do. what to do. what to do.

Tuesday, 7 September

To Do

  • Make repetitive sequence (MPR)
  • Make parts(for flush end ligation)
    • Annealing
    • Restriction Enzyme Digestion
    • Ethanol precipitation

(8)

  • Ligation

(7)(8)(10)(5)

  • transformation

Results

MPR
  • Electrophoresis results below indicated that sample E and F are proper for extracting.

Kyoto MPR 0907.png

Make parts(for flush end ligation)
Annealing product(8) conc.
NO.Sample nameconc./(ng/ul)
1annealing sample1.8
  • This indicated that annealing product was not obtained.
Transformation
  • (5)A,(5)B:Colonies were observed.
  • (7)A,(7)B,(8),(10)A,(10)B:No colony was observed.

Wednesday, 8 September

To Do

  • MPR products (further to 7 Sept.)
    • Gel extraction
    • Measure concentration
  • Make repetitive sequence (MPR)
  • (1)(4)(5)
    • Colony PCR
    • Electrophoresis
    • Miniprep and make master plates

Results

MPR products (further to 7 Sept.)

Kyoto MPR 0908.png

MPR product conc.
NO.band nameconc.
1117.4
2219.0
3318.4
4411.6
5517.9
Make repetitive sequence (MPR)
  • Gel extraction and Measured concentration.
MPR product conc.
NO.sample length /bpconc.
1-100021.5
21000-200042.3
32000-500020.5
45000+14.1

Kyoto MPR 0908 2.png

Colony PCR of (1),(4),(5)

Kyoto 0907 PCR.png

  • This result indicates that each part were inserted properly.

Thursday, 9 September

To Do

  • PCR products(1)(4)(5)(0908)
    • Gel extraction
    • Measure concentration (See lab note on 8 Sept.)
  • PCR product(5)
    • Miniprep and make master plates
  • (7)(8)(10)
    • Ligation
    • Transformation
  • Make parts(for flush end ligation) retry
    • Annealing
    • Restriction Enzyme Digestion
    • Ethanol precipitation

Results

(7),(8),(10)

We failed in the transformation 0907 because of the plasmid we used. We tried again using plasmid we purified anew.

  • PSB1A2(ES) conc.: 24(ng/ul)

After transformation, colonies were observed in all plates.

Make parts(for flush end ligation) retry
Annealing product conc./(ng/ul)
NO.sample nameconc.
1(11)49.8
2(11)'47.5

Friday, 10 August

To Do

  • (7)(8)(10)
    • Colony PCR
    • Electrophoresis
  • PCR

Results

(7)(8)(10)

Inserted properly

  • (7)A①③,B②
  • (8)①
  • (10)A①②③,B②③

Kyoto 0910 PCR.png This result indicates that some samples were inserted properly, though others were self ligated.

PCR

Kyoto 0910 PCR 2.png This indicates PCR was achieved.

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