Team:Kyoto/Notebook

From 2010.igem.org

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{{:Team:Kyoto/Header}}
{{:Team:Kyoto/Header}}
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==Index==
 
==Notebook==
==Notebook==
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<div class="note">
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===Notebooks===
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===Tuesday, July 20===
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* [[Team:Kyoto/Notebook1|Notebook1]]: Construction for Lysisbox.
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By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
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* [[Team:Kyoto/Notebook2|Notebook2]]: Measurement of R0011.
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====1. Solubilization of antibiotics.====
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* [[Team:Kyoto/Notebook3|Notebook3]]: Measurement of Lysis Cassette and Lysisbox etc.
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* for Ampicillin(Amp): add 1.0g Amp to 20ml MilliQ (final concentration is 50mg/ml).
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* for Kanamycin(kan): add 0.5g Kan to 10ml MilliQ (final concentration is 50mg/ml).
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* Dispense 1.1ml of the solution into 1.5ml tubes.
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* Store in the freezer (-20&#x2103;).
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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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{| class="experiments"
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!Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
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-
|-
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|<partinfo>J23100</partinfo>||1-18-C||1||20||21||rowspan="7"|LB (Amp+)||rowspan="8"|At 37&#x2103; 7/20 20:50 - 7/21 17:00||○
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|-
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|<partinfo>J23105</partinfo>||1-18-M||1||20||21||○
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|-
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|<partinfo>J23116</partinfo>||1-20-M||1||20||21||○
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|-
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|<partinfo>R0011</partinfo>||1-6-G||1||20||21||○
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|-
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|<partinfo>E0840</partinfo>||1-12-O||1||20||21||○
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|-
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|<partinfo>J06702</partinfo>||2-8-E||1||20||21||○
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|-
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|<partinfo>pSB4K5</partinfo>||1-5-G||1||20||21||×
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|-
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|<partinfo>B0015</partinfo>||1-23-L||1||20||21||LB (Kanamycin+)||×
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|}
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* "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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* 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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* So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".
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</div>
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<div class="note">
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[[#top-section|^Top]]
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===Wednesday, July 21===
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By: Wataru, Ken, Makoto, Takuya Yamamoto
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====1. Culture plates in which colonies was observed at 37&#x2103; from 07/21 20:50 to 07/22 17:00.====
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====2. Make a master plate of the above plates.====
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====3. Retry [[Team:Kyoto/Protocols#Transformation|Transformation]] of iGEM Parts.====
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{| class="experiments"
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!Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
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|-
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|<partinfo>pSB4K5</partinfo>||1-5-G||1||20||21||rowspan="2"|LB (Kanamycin+)||rowspan="2"|At 37℃ 7/21 20:50 - 7/22 16:30||○
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|-
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|<partinfo>B0015</partinfo>||1-23-L||1||20||21||○
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|}
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====4. [[Team:Kyoto/Protocols#PCR|PCR]] for S-R-Rz/Rz1 and S====
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* Dilute &lambda;DNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template &lambda;DNA was 5ng/µl.
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{| class="experiments"
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!No.||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||TemplateDNA (5ng/µl)||Primer S-R-Rz/Rz1 Forward (10µM)||Primer S-R-Rz/Rz1 Reverse (10µM)||Primer S Reverse (10µM)||KOD Plus ver.2||Total
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|-
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|1||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
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|-
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|2||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
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|-
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|3||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
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|-
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|4||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
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|-
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|5||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
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|-
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|6||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
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|-
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|7||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
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|-
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|8||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
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|}
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* Forward Primer of S-R-Rz/Rz1 and S is common.
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* PCR condition : 94&#x2103; x 2min, (98&#x2103; x 10sec, 55&#x2103; x 30sec, 68&#x2103; x 1min) x 30cycles, 4&#x2103; forever.
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</div>
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<div class="note">
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===Other Information===
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===Thursday, July 22===
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* [[Team:Kyoto/Protocols|Protocols]]: Protocols of each experiment such as Polymerase Chain Reaction (PCR), Restriction Digestion, Ligation, Transformation.
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By: Wataru
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* [[Team:Kyoto/Materials|Materials]]: Strains, DNA, and Primers.
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====1. [[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of the PCR products for 40min.====
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* [[Team:Kyoto/Parts|Parts]]: Construction of each part and BioBrick Parts used in our project.
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[[Image:KyotoEXP100722-1.png|right]]
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* '''Discussion'''
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* Length of S and S-R-Rz/Rz1 is 370bp, 1300bp, so PCR succeeded.
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====2. [[Team:Kyoto/Protocols#Miniprep|Miniprep]] of iGEM Parts.====
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{| class="experiments"
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!Name||Concentration(ng/&micro;l)
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|-
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|<partinfo>J23100</partinfo>||18.5
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|-
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|<partinfo>J23105</partinfo>||12.5
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|-
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|<partinfo>J23116</partinfo>||14.6
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|-
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|<partinfo>R0011</partinfo>||8.6
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|-
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|<partinfo>E0840</partinfo>||12.1
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|-
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|<partinfo>J06702</partinfo>||14.7
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|}
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* '''Discussion'''
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* The concentration of all samples was very week. Probably our shaking incubation was week.
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====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.====
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</div>
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<div class="note">
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[[#top-section|^Top]]
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===Friday, July 23===
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By: Wataru, Tomo, Makoto
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====1. [[Team:Kyoto/Protocols#Miniprep|Miniprep]] of iGEM Parts.====
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{| class="experiments"
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!Name||Concentration(ng/&micro;l)
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|-
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|<partinfo>pSB4K5</partinfo>||79.2
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|-
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|<partinfo>B0015</partinfo>||-
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|}
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* '''Discussion'''
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* We lost <partinfo>B0015</partinfo> by our mistake.
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* The concentration of <partinfo>pSB4K5</partinfo> is high, so this condition of shaking incubation is moderate.
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====2. Picked up 1, 3, 5, 7 of the products of PCR, and purified by PCR-purification.====
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{| class="experiments"
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!Sample||Concentration (ng/&micro;l)||New Name||
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|-
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|1||18.6||-
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|-
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|3||77.6||S<sub>1</sub>
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|-
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|5||33.6||-
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|-
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|7||65.4||S<sub>2</sub>
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|}
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* '''Discussion'''
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* The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.
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====3. Retry of PCR of S-R-Rz/Rz1.====
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{| class="experiments"
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!Sample||Water||25mmol/l MgSO4||2mmol/l dNTPs||10×Buffer for KOD plus ver.2||Template DNA (5ng/&micro;l)||Primer S-R-Rz/Rz1 Forward (10&micro;mol/l)||Primer S-R-Rz/Rz1 Reverse (10&micro;mol/l)||KOD plus ver.2||Total
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|-
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|1||28&micro;l||3||5||5||5||1.5||1.5||1||50
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|-
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|2||28||3||5||5||5||1.5||1.5||1||50
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|-
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|3||26.5||4.5||5||5||5||1.5||1.5||1||50
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|-
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|4||26.5||4.5||5||5||5||1.5||1.5||1||50
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|-
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|5||25||6||5||5||5||1.5||1.5||1||50
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|-
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|6||25||6||5||5||5||1.5||1.5||1||50
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|}
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* PCR condition : 94&#x2103; x 2min, (98&#x2103; x 10sec, 55&#x2103; x 30sec, 68&#x2103; x 1min) x 30cycles, 4&#x2103; forever.
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====4. Digested <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, PstI to check function of our Restriction enzymes.====
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{| class="experiments"
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!Sample||10xBuffer||BSA||Enzyme||MilliQ||Total||Incubation
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|-
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|1||5&micro;l||1||''EcoR''I 0.1||3.6||10||rowspan="5"|At 37&#x2103; 7/23 18:00 - 7/23 18:30
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|-
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|2||5||1||''Xba''I 0.1||3.6||10
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|-
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|3||5||1||''Spe''I 0.1||3.6||10
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|-
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|4||5||1||''Pst''I 0.1||3.6||10
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|-
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|5||5||1||-||3.7||10
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|}
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====5. Electrophoresis of above sample for 35min.====
 
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[[Image:KyotoExp100723-1.png|right]]
 
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* '''Discussion'''
 
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* 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.
 
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====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.====
 
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{| class="experiments"
 
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!Sample||10×Buffer||Enzyme 1||Enzyme 2||MilliQ||Total||Incubation
 
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|-
 
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|S<sub>1</sub>||11&micro;l||5||''EcoR''I 0.2||''Spe''I 0.2||33.6||50||rowspan="3"|At 37&#x2103; for 2h
 
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|-
 
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|S<sub>2</sub>||11||5||''EcoR''I 0.2||''Spe''I 0.2||33.6||50
 
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|-
 
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|<partinfo>E0840</partinfo>(GFP)||45||5||''EcoR''I 0.2||''Xba''I 0.2||0||50
 
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|}
 
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* After PCR purification, evaporated them and diluted 3ul.
 
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====7. Ligated over night.====
 
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{| class="experiments"
 
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!Sample||Vector||Insert||Ligation High||Total
 
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|-
 
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|S-GFP<sub>1</sub>||<partinfo>E0840</partinfo> 0.5&micro;l||S<sub>1</sub> 0.5||1||2
 
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|-
 
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|S-GFP<sub>2</sub>||<partinfo>E0840</partinfo> 0.5||S<sub>2</sub> 0.5||1||2
 
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|}
 
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</div>
 
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<div class="note">
 
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===Monday, July 26===
 
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By: Wataru, Tomonori, Makoto
 
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====1. Electrophoresis of PCR products====
 
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[[Image:KyotoExp100726-1.png|right]]
 
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* '''Discussion'''
 
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* At the condition 4 (4.5&micro;l MgSO4) and 6 (6&micro;l MgSO4), S-R-Rz/Rz1 is amplified very much. So we decided to use them.
 
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====2. PCR purification====
 
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{| class="experiments"
 
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!Sample||Concentration (ng/&micro;l)||New Name
 
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|-
 
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|4||51.6||
 
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|-
 
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|5||59.3||
 
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|-
 
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|6||59.6||
 
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|}
 
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====3. Transformation of iGEM Parts====
 
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{| class="experiments"
 
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!Name||Well||Sample (&micro;l)||Competent Cell (&micro;l)||Total (&micro;l)||Plate||Incubation||Result
 
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|-
 
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|||1-12-M||1||20||21||LB (Amplicillin+)||rowspan="3"|At 37&#x2103; 7/26 - 7/27||×
 
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|-
 
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|||2-17-F||1||20||21||rowspan="2"|LB (Kanamycin+)||×
 
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|-
 
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|||1-5-E||1||20||21||×
 
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|}
 
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====4. Culture of 1-6-G, 1-12-O, and 1-23-L=====
 
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</div>
 
----
----

Latest revision as of 11:33, 27 October 2010

Contents

Notebook

Notebooks

^Top

Other Information

  • Protocols: Protocols of each experiment such as Polymerase Chain Reaction (PCR), Restriction Digestion, Ligation, Transformation.
  • Materials: Strains, DNA, and Primers.
  • Parts: Construction of each part and BioBrick Parts used in our project.

^Top

Retrieved from "http://2010.igem.org/Team:Kyoto/Notebook"