Team:Kyoto/Notebook

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Revision as of 13:39, 8 September 2010

Contents

Index

Notebook

Tuesday, July 20

By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto

1. Solubilization of antibiotics.

  • for Ampicillin(Amp): add 1.0g Amp to 20ml MilliQ (final concentration is 50mg/ml).
  • for Kanamycin(kan): add 0.5g Kan to 10ml MilliQ (final concentration is 50mg/ml).
  • Dispense 1.1ml of the solution into 1.5ml tubes.
  • Store in the freezer (-20℃).

2. Making plates for LB (Amp+) and LB (Kan+).

3. Transformation of iGEM Parts.

NameWellSample (µl)Competent Cells (µl)Total (µl)PlateIncubationResult
<partinfo>J23100</partinfo>1-18-C12021LB (Amp+)At 37℃ 7/20 20:50 - 7/21 17:00○
<partinfo>J23105</partinfo>1-18-M12021○
<partinfo>J23116</partinfo>1-20-M12021○
<partinfo>R0011</partinfo>1-6-G12021○
<partinfo>E0840</partinfo>1-12-O12021○
<partinfo>J06702</partinfo>2-8-E12021○
<partinfo>pSB4K5</partinfo>1-5-G12021×
<partinfo>B0015</partinfo>1-23-L12021LB (Kanamycin+)×
  • "1-18-C" means well 18C in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
  • Discussion
  • A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Ampicillin+).
  • And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance.
  • So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".

Wednesday, July 21

By: Wataru, Ken, Makoto, Takuya Yamamoto

1. Culture plates in which colonies was observed at 37℃ from 07/21 20:50 to 07/22 17:00.

2. Make a master plate of the above plates.

3. Retry Transformation of iGEM Parts.

NameWellSample (µl)Competent Cells (µl)Total (µl)PlateIncubationResult
<partinfo>pSB4K5</partinfo>1-5-G12021LB (Kanamycin+)At 37℃ 7/21 20:50 - 7/22 16:30○
<partinfo>B0015</partinfo>1-23-L12021○

4. PCR for S-R-Rz/Rz1 and S

  • Dilute λDNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template λDNA was 5ng/µl.
No.Water25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2TemplateDNA (5ng/µl)Primer S-R-Rz/Rz1 Forward (10µM)Primer S-R-Rz/Rz1 Reverse (10µM)Primer S Reverse (10µM)KOD Plus ver.2Total
128µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
228µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
328µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
428µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
528µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
628µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
728µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
828µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
  • Forward Primer of S-R-Rz/Rz1 and S is common.
  • PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.

Thursday, July 22

By: Wataru

1. Electrophoresis of the PCR products for 40min.

File:KyotoEXP100722-1.png
  • Discussion
  • Length of S and S-R-Rz/Rz1 is 370bp, 1300bp, so PCR succeeded.

2. Miniprep of iGEM Parts.

NameConcentration(ng/µl)
<partinfo>J23100</partinfo>18.5
<partinfo>J23105</partinfo>12.5
<partinfo>J23116</partinfo>14.6
<partinfo>R0011</partinfo>8.6
<partinfo>E0840</partinfo>12.1
<partinfo>J06702</partinfo>14.7
  • Discussion
  • The concentration of all samples was very week. Probably our shaking incubation was week.

3. Culture plates and make master plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo> from 7/22 17:00 to 7/23 10:00.

Friday, July 23

By: Wataru, Tomo, Makoto

1. Miniprep of iGEM Parts.

NameConcentration(ng/µl)
<partinfo>pSB4K5</partinfo>79.2
<partinfo>B0015</partinfo>-
  • Discussion
  • We lost <partinfo>B0015</partinfo> by our mistake.
  • The concentration of <partinfo>pSB4K5</partinfo> is high, so this condition of shaking incubation is moderate.

2. Picked up 1, 3, 5, 7 of the products of PCR, and purified by PCR-purification.

SampleConcentration (ng/µl)New Name
118.6-
377.6S1
533.6-
765.4S2
  • Discussion
  • The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.

3. Retry of PCR of S-R-Rz/Rz1.

SampleWater25mmol/l MgSO42mmol/l dNTPs10×Buffer for KOD plus ver.2Template DNA (5ng/µl)Primer S-R-Rz/Rz1 Forward (10µmol/l)Primer S-R-Rz/Rz1 Reverse (10µmol/l)KOD plus ver.2Total
128µl35551.51.5150
22835551.51.5150
326.54.55551.51.5150
426.54.55551.51.5150
52565551.51.5150
62565551.51.5150
  • PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.

4. Digested <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, PstI to check function of our Restriction enzymes.

Sample10xBufferBSAEnzymeMilliQTotalIncubation
15µl1EcoRI 0.13.610At 37℃ 7/23 18:00 - 7/23 18:30
251XbaI 0.13.610
351SpeI 0.13.610
451PstI 0.13.610
551-3.710

5. Electrophoresis of above sample for 35min.

KyotoExp100723-1.png
  • Discussion
  • Comparison to sample 5(control, circular DNA), the bands of sample 1, 2, 3, 4 was shifted. The DNA of sample 1, 2, 3, 4 was linearized by Restriction enzymes. So, our restriction enzymes work correctly.

6. To insert S gene to GFP, we digested the PCR products of S gene by EcoRi and SpeI, and GFP by EcoRl and XbaI.

Sample10×BufferEnzyme 1Enzyme 2MilliQTotalIncubation
S111µl5EcoRI 0.2SpeI 0.233.650At 37℃ for 2h
S2115EcoRI 0.2SpeI 0.233.650
<partinfo>E0840</partinfo>(GFP)455EcoRI 0.2XbaI 0.2050
  • After PCR purification, evaporated them and diluted 3ul.

7. Ligated over night.

SampleVectorInsertLigation HighTotal
S-GFP1<partinfo>E0840</partinfo> 0.5µlS1 0.512
S-GFP2<partinfo>E0840</partinfo> 0.5S2 0.512

Monday, July 26

By: Wataru, Tomonori, Makoto

1. Electrophoresis of PCR products

KyotoExp100726-1.png
  • Discussion
  • At the condition 4 (4.5µl MgSO4) and 6 (6µl MgSO4), S-R-Rz/Rz1 is amplified very much. So we decided to use them.

2. PCR purification

SampleConcentration (ng/µl)New Name
451.6
559.3
659.6

3. Transformation of iGEM Parts

NameWellSample (µl)Competent Cell (µl)Total (µl)PlateIncubationResult
1-12-M12021LB (Amplicillin+)At 37℃ 7/26 - 7/27×
2-17-F12021LB (Kanamycin+)×
1-5-E12021×

4. Culture of 1-6-G, 1-12-O, and 1-23-L

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