Team:Kyoto/Notebook

From 2010.igem.org

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| By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
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*By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
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*Category: Antibiotic, Transformation
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|(1) Solubilized of antibiotics, Ampicillin and Kanamycin as the following.
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| (1) Solubilization of antibiotics.
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* 1. Mix the following (final concentration is 50mg/ml).
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| For Ampicillin(Amp): add 1.0g Amp to 20ml MilliQ (Final concentration is 50mg/ml).  
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** For Ampicillin: 1g Ampicillin and 20ml MilliQ.
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| For Kanamycin(Kan): add 0.5g Kan to 10ml MilliQ (Final concentration is 50mg/ml).
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** For Kanamycin: 0.5g Kanamycin and 10ml MilliQ.
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| Dispense 1.1ml of the solution into 1.5ml tubes.
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* 2. Dispense 1.1ml of the mixture into 1.5ml tubes.
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| Store in the freezer (-20℃).
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* 3. Store in the freezer (-20℃).
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|(2) [[Team:Kyoto/Protocols#Making_Plate|Made plates]] for LB (Ampicillin+) and LB (Kanamycin+).
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| (2) Making plates for LB (Amp+) and LB (Kan+).
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|(3) [[Team:Kyoto/Protocols#Transformation|Transformation]] of iGEM Parts.  
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| (3) [[Team:Kyoto/Protocols#Transformation|Transformation]] of iGEM Parts.
{| class="experiments"
{| class="experiments"
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!Name||Well<sup>*1</sup>||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
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!Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
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|<partinfo>J23100</partinfo>||1-18-C||1||20||21||rowspan="7"|LB (Ampicillin+)||rowspan="8"|At 37&#x2103; 7/20 20:50 - 7/21 17:00||○
|<partinfo>J23100</partinfo>||1-18-C||1||20||21||rowspan="7"|LB (Ampicillin+)||rowspan="8"|At 37&#x2103; 7/20 20:50 - 7/21 17:00||○
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|<partinfo>B0015</partinfo>||1-23-L||1||20||21||LB (Kanamycin+)||×
|<partinfo>B0015</partinfo>||1-23-L||1||20||21||LB (Kanamycin+)||×
|}
|}
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* *1 "1-18-C" means well 18C in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
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| *1 "1-18-C" means well 18C in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
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* '''Discussion'''
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| '''Discussion'''
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* A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Ampicillin+).
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| A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Ampicillin+).
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* And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance.
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| And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance.
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* So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".
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| So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".
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|}
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Revision as of 16:06, 6 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 (Ampicillin+)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 "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
  • Category: Transformation, PCR, Lysis Cassette
(1) Cultured plates in which colonies was observed at 37℃ from 07/21 20:50 to 07/22 17:00.
(2) Made a master plate of the above plates.
(3) Retried Transformation of iGEM Parts.
NameWell*1Sample (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○
  • *1 "1-5-G" means well 5G in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
(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 22, July
  • By: Wataru
  • Category: Lysis Cassette, parts
(1) Electrophoresis of the PCR products for 40min.
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 23, July
  • By: Wataru, Tomo, Makoto
  • Category:
(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.
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
  • Category:
(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.
PCR purification
SampleConcentration (ng/µl)New Name
451.6
559.3
659.6
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×
Culture of 1-6-G, 1-12-O, and 1-23-L
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