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| | {{:Team:Kyoto/Header}} | | {{:Team:Kyoto/Header}} |
| - | ==Index==
| |
| | ==Notebook== | | ==Notebook== |
| | + | ===Notebooks=== |
| | + | * [[Team:Kyoto/Notebook1|Notebook1]]: Construction for Lysisbox. |
| | + | * [[Team:Kyoto/Notebook2|Notebook2]]: Measurement of R0011. |
| | + | * [[Team:Kyoto/Notebook3|Notebook3]]: Measurement of Lysis Cassette and Lysisbox etc. |
| | | | |
| - | <div class="note">
| + | [[#top-section|^Top]] |
| | | | |
| - | ===Tuesday, July 20=== | + | ===Other Information=== |
| | + | * [[Team:Kyoto/Protocols|Protocols]]: Protocols of each experiment such as Polymerase Chain Reaction (PCR), Restriction Digestion, Ligation, Transformation. |
| | + | * [[Team:Kyoto/Materials|Materials]]: Strains, DNA, and Primers. |
| | + | * [[Team:Kyoto/Parts|Parts]]: Construction of each part and BioBrick Parts used in our project. |
| | | | |
| - | By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
| + | [[#top-section|^Top]] |
| | | | |
| - | ====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. [[Team:Kyoto/Protocols#Transformation|Transformation]] of iGEM Parts.====
| |
| - |
| |
| - | {| class="experiments"
| |
| - | !Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
| |
| - | |-
| |
| - | |<partinfo>J23100</partinfo>||1-18-C||1||20||21||rowspan="7"|LB (Amp+)||rowspan="8"|At 37℃ 7/20 20:50 - 7/21 17:00||○
| |
| - | |-
| |
| - | |<partinfo>J23105</partinfo>||1-18-M||1||20||21||○
| |
| - | |-
| |
| - | |<partinfo>J23116</partinfo>||1-20-M||1||20||21||○
| |
| - | |-
| |
| - | |<partinfo>R0011</partinfo>||1-6-G||1||20||21||○
| |
| - | |-
| |
| - | |<partinfo>E0840</partinfo>||1-12-O||1||20||21||○
| |
| - | |-
| |
| - | |<partinfo>J06702</partinfo>||2-8-E||1||20||21||○
| |
| - | |-
| |
| - | |<partinfo>pSB4K5</partinfo>||1-5-G||1||20||21||×
| |
| - | |-
| |
| - | |<partinfo>B0015</partinfo>||1-23-L||1||20||21||LB (Kanamycin+)||×
| |
| - | |}
| |
| - |
| |
| - | 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".
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <div class="note">
| |
| - | ===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 [[Team:Kyoto/Protocols#Transformation|Transformation]] of iGEM Parts.====
| |
| - |
| |
| - | {| class="experiments"
| |
| - | !Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
| |
| - | |-
| |
| - | |<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||○
| |
| - | |-
| |
| - | |<partinfo>B0015</partinfo>||1-23-L||1||20||21||○
| |
| - | |}
| |
| - |
| |
| - | ====4. [[Team:Kyoto/Protocols#PCR|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.
| |
| - |
| |
| - | {| class="experiments"
| |
| - | !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
| |
| - | |-
| |
| - | |1||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
| |
| - | |-
| |
| - | |2||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
| |
| - | |-
| |
| - | |3||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
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| - | |-
| |
| - | |4||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
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| - | |-
| |
| - | |5||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
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| - | |-
| |
| - | |6||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
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| - | |-
| |
| - | |7||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
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| - | |-
| |
| - | |8||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
| |
| - | |}
| |
| - |
| |
| - | Forward Primer of S-R-Rz/Rz1 and S is common.
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| - |
| |
| - | PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <div class="note">
| |
| - |
| |
| - | ===Thursday, July 22===
| |
| - |
| |
| - | By: Wataru
| |
| - |
| |
| - | ====1. [[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of the PCR products for 40min.====
| |
| - |
| |
| - | [[Image:KyotoEXP100722-1.png|right]]
| |
| - |
| |
| - | Length of S and S-R-Rz/Rz1 is 370bp, 1300bp, so PCR succeeded.
| |
| - |
| |
| - | ====2. [[Team:Kyoto/Protocols#Miniprep|Miniprep]] of iGEM Parts.====
| |
| - |
| |
| - | {| class="experiments"
| |
| - | !Name||Concentration(ng/µl)
| |
| - | |-
| |
| - | |<partinfo>J23100</partinfo>||18.5
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| - | |-
| |
| - | |<partinfo>J23105</partinfo>||12.5
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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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| - | |-
| |
| - | |<partinfo>E0840</partinfo>||12.1
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| - | |-
| |
| - | |<partinfo>J06702</partinfo>||14.7
| |
| - | |}
| |
| - |
| |
| - | 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.====
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <div class="note">
| |
| - |
| |
| - | ===Friday, July 23===
| |
| - |
| |
| - | By: Wataru, Tomo, Makoto
| |
| - |
| |
| - | ====1. [[Team:Kyoto/Protocols#Miniprep|Miniprep]] of iGEM Parts.====
| |
| - | {| class="experiments"
| |
| - |
| |
| - | !Name||Concentration(ng/µl)
| |
| - | |-
| |
| - | |<partinfo>pSB4K5</partinfo>||79.2
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| - | |-
| |
| - | |<partinfo>B0015</partinfo>||-
| |
| - | |}
| |
| - |
| |
| - | 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.====
| |
| - |
| |
| - | {| class="experiments"
| |
| - | !Sample||Concentration (ng/µl)||New Name||
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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>
| |
| - | |}
| |
| - |
| |
| - | 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.====
| |
| - |
| |
| - | {| class="experiments"
| |
| - | !Sample||Water||25mmol/l MgSO4||2mmol/l dNTPs||10×Buffer for KOD plus ver.2||Template DNA (5ng/µl)||Primer S-R-Rz/Rz1 Forward (10µmol/l)||Primer S-R-Rz/Rz1 Reverse (10µmol/l)||KOD plus ver.2||Total
| |
| - | |-
| |
| - | |1||28µl||3||5||5||5||1.5||1.5||1||50
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| - | |-
| |
| - | |2||28||3||5||5||5||1.5||1.5||1||50
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| - | |-
| |
| - | |3||26.5||4.5||5||5||5||1.5||1.5||1||50
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| - | |-
| |
| - | |4||26.5||4.5||5||5||5||1.5||1.5||1||50
| |
| - | |-
| |
| - | |5||25||6||5||5||5||1.5||1.5||1||50
| |
| - | |-
| |
| - | |6||25||6||5||5||5||1.5||1.5||1||50
| |
| - | |}
| |
| - |
| |
| - | 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.====
| |
| - |
| |
| - | {| class="experiments"
| |
| - | !Sample||10xBuffer||BSA||Enzyme||MilliQ||Total||Incubation
| |
| - | |-
| |
| - | |1||5µl||1||''EcoR''I 0.1||3.6||10||rowspan="5"|At 37℃ 7/23 18:00 - 7/23 18:30
| |
| - | |-
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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
| |
| - | |}
| |
| - |
| |
| - | ====5. Electrophoresis of above sample for 35min.====
| |
| - |
| |
| - | [[Image:KyotoExp100723-1.png|right]]
| |
| - |
| |
| - | 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.====
| |
| - |
| |
| - | {| class="experiments"
| |
| - | !Sample||10×Buffer||Enzyme 1||Enzyme 2||MilliQ||Total||Incubation
| |
| - | |-
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| - | |S<sub>1</sub>||11µl||5||''EcoR''I 0.2||''Spe''I 0.2||33.6||50||rowspan="3"|At 37℃ for 2h
| |
| - | |-
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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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| - |
| |
| - | After PCR purification, evaporated them and diluted 3ul.
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| - |
| |
| - | ====7. Ligated over night.====
| |
| - |
| |
| - | {| class="experiments"
| |
| - | !Sample||Vector||Insert||Ligation High||Total
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| - | |-
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| - | |S-GFP<sub>1</sub>||<partinfo>E0840</partinfo> 0.5µl||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
| |
| - | |}
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <div class="note">
| |
| - |
| |
| - | ===Monday, July 26===
| |
| - |
| |
| - | By: Wataru, Tomonori, Makoto
| |
| - |
| |
| - | ====1. Electrophoresis of PCR products====
| |
| - |
| |
| - | [[Image:KyotoExp100726-1.png|right]]
| |
| - |
| |
| - | 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====
| |
| - |
| |
| - | {| class="experiments"
| |
| - | !Sample||Concentration (ng/µl)||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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| - | |-
| |
| - | |6||59.6||
| |
| - | |}
| |
| - |
| |
| - | ====3. Transformation of iGEM Parts====
| |
| - |
| |
| - | {| class="experiments"
| |
| - | !Name||Well||Sample (µl)||Competent Cell (µl)||Total (µl)||Plate||Incubation||Result
| |
| - | |-
| |
| - | |||1-12-M||1||20||21||LB (Amplicillin+)||rowspan="3"|At 37℃ 7/26 - 7/27||×
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| - | |-
| |
| - | |||2-17-F||1||20||21||rowspan="2"|LB (Kanamycin+)||×
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| - | |-
| |
| - | |||1-5-E||1||20||21||×
| |
| - | |}
| |
| - |
| |
| - | ====4. Culture of 1-6-G, 1-12-O, and 1-23-L====
| |
| - |
| |
| - | </div>
| |
| | ---- | | ---- |
| - |
| |
| - | ===Monday, July 26===
| |
| - | By: Wataru, Tomonari, Makoto
| |
| - |
| |
| - | ====Electrophoresis of PCR products====
| |
| - |
| |
| - | 3①3②4.5①4.5②6①6②
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| - |
| |
| - | [[image : KyotoExp100726-1.png]]
| |
| - |
| |
| - | Discussion
| |
| - |
| |
| - | At the condition 4.5② and 6②、S-R-Rz is amplified very much. So we decided to use them inexperience.
| |
| - |
| |
| - | ====PCR purification====
| |
| - | {|class="wikitable"
| |
| - | !Sample number||Concentration(ng/µL)
| |
| - | |-
| |
| - | |4.5②||51.6
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| - | |-
| |
| - | |6①||59.3
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| - | |-
| |
| - | |6②||59.6
| |
| - | |}
| |
| - |
| |
| - | ====Transformation====
| |
| - | {|class="wikitable"
| |
| - | !Sample||Conc(/µL)||Sample Volume(µL)||Competent Cell(µL)||Total||Plate||Incubation
| |
| - | |-
| |
| - | |1-12-M||-||1||20||21||LB amp||rowspan="3"|7/26~7/27
| |
| - | |-
| |
| - | |2-17-F||-||1||20||21||rowspan="2"|LB kan
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| - | |-
| |
| - | |1-5-E||-||1||20||21
| |
| - | |}
| |
| - |
| |
| - | ====Culture====
| |
| - |
| |
| - | 1-6-G, 1-12-O, 1-23-L
| |
| - |
| |
| - | ===Tuesday, July 27===
| |
| - | By: Wataru, Tomo, Kazuya, Ken, Naoi
| |
| - |
| |
| - | Result of transformation
| |
| - | {|class="wikitable"
| |
| - | |1-12-M||rowspan="3"|No colony
| |
| - | |-
| |
| - | |1-5-E
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| - | |-
| |
| - | |2-17-F
| |
| - | |}
| |
| - |
| |
| - | ====Colony of PCR of S-E840====
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| - |
| |
| - | To check that S GFP is correctly inserted, we did colony PCR.
| |
| - |
| |
| - | Gel: Agarose
| |
| - | Time: 35min
| |
| - | Voltage: 100V
| |
| - | Maker: 1K 100
| |
| - | 1～6 S-GFP①
| |
| - | 7～13 S-GFP②
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| - | Posi E840（about 900bp）
| |
| - | Nega None
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| - |
| |
| - | 1K 1 2 3 4 5 6 7 8 9 10 11 12 13 posi nega 100
| |
| - | [[image:KyotoExp100727.png]]
| |
| - |
| |
| - | As a result, 1,3,5,6,11,12,13 are inserted S gene correctly.
| |
| - | So, we decided to use 6 as S-E840① and 11 as S-E840②.
| |
| - |
| |
| - | ====Miniprep====
| |
| - | {|class="wikitable"
| |
| - | !Sample number||Concentration(ng/µL)
| |
| - | |-
| |
| - | |1-6-G||26.9
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| - | |-
| |
| - | |1-23-L||120.0
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| - | |-
| |
| - | |1-12-O||120.1
| |
| - | |}
| |
| - |
| |
| - | ====RE====
| |
| - | {|class="wikitable"
| |
| - | !||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
| |
| - | |-
| |
| - | |1-23-L||30||5||0.5||rowspan="3"|EcoRI||0.4||rowspan="3"|XbaI||0.3||13.7||50
| |
| - | |-
| |
| - | |4.5②||40||5||0.5||0.4||0.4||3.8||50
| |
| - | |-
| |
| - | |6②||40||5||0.5||0.4||0.4||3.8||50
| |
| - | |}
| |
| - | Incubate 37℃ 16:45~18:00
| |
| - |
| |
| - | ====Ligation====
| |
| - |
| |
| - | ====Transformation====
| |
| - |
| |
| - | ===Wednesday, July 28===
| |
| - |
| |
| - | ====1. Result of Transformation====
| |
| - | {|class="wikitable"
| |
| - | |SRRz①-DT||rowspan="2"|Many colonies
| |
| - | |-
| |
| - | |SRRz②-DT
| |
| - | |}
| |
| - |
| |
| - | ====2. Deletion PCR====
| |
| - | To delete functional domain of S gene, we did deletion PCR.
| |
| - |
| |
| - | =====Miniprep=====
| |
| - | {|class="wikitable"
| |
| - | !Sample number||Concentration(ng/µ)
| |
| - | |-
| |
| - | |S-E840①||95.5
| |
| - | |-
| |
| - | |S-E840②||98.6
| |
| - | |}
| |
| - |
| |
| - | Diluted S-GFP① and S-GFP② 20 times with water, and used as template DNA.
| |
| - |
| |
| - | =====Deletion PCR=====
| |
| - |
| |
| - | To delete functional region of S gene, we did deletion PCR.
| |
| - |
| |
| - | {|class="wikitable"
| |
| - | !||Water||25mM MgSO4||2mM dNTPs||10x buffer for KOD Plus ver.2||Primer Deletion F(10µM)||Primer Deletion R(10µM)||Template S-E840①||Template S-E840②||KOD Plus ver.2||Total
| |
| - | |-
| |
| - | |Δ1-1||28||3||5||5||1.5||1.5||5||-||1||50
| |
| - | |-
| |
| - | |Δ1-2||28||3||5||5||1.5||1.5||5||-||1||50
| |
| - | |-
| |
| - | |Δ2-1||28||3||5||5||1.5||1.5||-||5||1||50
| |
| - | |}
| |
| - |
| |
| - | PCR program
| |
| - | {|class="wikitable"
| |
| - | |94℃||2min||
| |
| - | |-
| |
| - | |98℃||10sec||rowspan="3"|35 cycles
| |
| - | |-
| |
| - | |55℃||30sec
| |
| - | |-
| |
| - | |-
| |
| - | |68℃||4min
| |
| - | |-
| |
| - | |4℃||forever||
| |
| - | |}
| |
| - |
| |
| - | ====3. RE====
| |
| - | To check function of our Restriction enzymes, we digested S-E840① and S-E-840② by DpnI.
| |
| - | {|class="wikitable"
| |
| - | !||Sample||fast digestion buffer||DpnI||MilliQ||Total
| |
| - | |-
| |
| - | |S-E840①||3||1||0.1||5.8||10
| |
| - | |-
| |
| - | |S-E840②||3||1||0.1||5.8||10
| |
| - | |}
| |
| - |
| |
| - | =====Electrophoresis=====
| |
| - | Gel: Agarose
| |
| - | Time: 35min
| |
| - | Voltage: 100V
| |
| - | Maker: 1K 100
| |
| - |
| |
| - | 1 1k marker 2 not digested S-E840① 3 not digested S-E840② 4 digested S-E840① 5 digested S-E840② 6 100bp marker
| |
| - |
| |
| - | 1k 1 2 3 4 100
| |
| - |
| |
| - | [[image:KyotoExp100728.png]]
| |
| - |
| |
| - | Discussion
| |
| - |
| |
| - | DpnI works correctly
| |
| - |
| |
| - | ===Thursday, July 29===
| |
| - |
| |
| - | ====RE====
| |
| - | {|class="wikitable"
| |
| - | !||Sample volume||Fastdigestion buffer||Enzyme 1||MilliQ||Total
| |
| - | |-
| |
| - | |Δ1-1||50||6||DpnI 0.2||3.8||60
| |
| - | |-
| |
| - | |Δ2-1||50||6||DpnI 0.2||3.8||60
| |
| - | |}
| |
| - | Incubate 7/29 9:40~7/29 11:00
| |
| - |
| |
| - | ====Ligation and Pospholylation====
| |
| - | {|class="wikitable"
| |
| - | !||Sample||MilliQ||Ligation High||T4 Kinase||Total
| |
| - | |-
| |
| - | |Δ1-1||2||7||5||1||15
| |
| - | |-
| |
| - | |Δ2-1||2||7||5||1||15
| |
| - | |}
| |
| - | Incubate 7/29 11:30~7/29 13:00
| |
| - |
| |
| - | ====Transformation====
| |
| - | {|class="wikitable"
| |
| - | !Sample||Conc(/µL)||Sample Volume(µL)||Competent Cell(µL)||Total||Plate||Incubation
| |
| - | |-
| |
| - | |Δ1-1||-||3||30||33||rowspan="2"|LB amp||rowspan="2"|7/29~7/30
| |
| - | |-
| |
| - | |Δ1-1||-||3||30||33
| |
| - | |}
| |
| - |
| |
| - | ===Friday, July 30===
| |
| - | Result of transformation of Δ1 and Δ2
| |
| - | Many colonies are observed.
| |
| - |
| |
| - | ===Monday, August 2===
| |
| - | By: Wataru, Ken
| |
| - |
| |
| - | ====1. Miniprep====
| |
| - |
| |
| - | {|class="wikitable"
| |
| - | !Sample number||Concentration(ng/µL)
| |
| - | |-
| |
| - | |Δ1||52.7
| |
| - | |-
| |
| - | |Δ2||54.4
| |
| - | |-
| |
| - | |Δ3||89.5
| |
| - | |-
| |
| - | |pSB4K5||50.7
| |
| - | |-
| |
| - | |LacP||18.6
| |
| - | |}
| |
| - |
| |
| - | ====2. PCR and RE of E240====
| |
| - |
| |
| - | E240 is very important parts to measure RPU of promoters in iGEM. However, we failed to transfect it to E.coli from parts kit of iGEM. So we decided to amplify this parts by PCR.
| |
| - |
| |
| - | {|class="wikitable"
| |
| - | !||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10µM)||Primer VR(10µM)||Template E240||KOD Pllus ver.2||Total
| |
| - | |-
| |
| - | |E240①||28||3||5||5||1.5||1.5||5||1||50
| |
| - | |-
| |
| - | |E240②||28||3||5||5||1.5||1.5||5||1||50
| |
| - | |}
| |
| - |
| |
| - | PCR program
| |
| - | {|class="wikitable"
| |
| - | |94℃||2min||
| |
| - | |-
| |
| - | |98℃||10sec||rowspan="3"|35 cycles
| |
| - | |-
| |
| - | |55℃||30sec
| |
| - | |-
| |
| - | |68℃||4min
| |
| - | |-
| |
| - | |4℃||forever||
| |
| - | |}
| |
| - |
| |
| - | ====Electrophoresis====
| |
| - |
| |
| - | ====PCR purification====
| |
| - | {|class="wikitable"
| |
| - | !Sample number||Concentration(ng/µL)
| |
| - | |-
| |
| - | |E240①||42.6
| |
| - | |-
| |
| - | |E240②||55.3
| |
| - | |}
| |
| - |
| |
| - | ====RE====
| |
| - | To insert E240 to pSB4K5 by 3A assembly, we digested the PCR products of E240 by XbaI and PstI
| |
| - | {|class="wikitable"
| |
| - | !||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
| |
| - | |-
| |
| - | |E240①X-P||30||5||0.5||XbaI||0.2||PstI||0.2||14.1||50
| |
| - | |-
| |
| - | |E240②X-P||30||5||0.5||XbaI||0.2||PstI||0.2||14.1||50
| |
| - | |}
| |
| - |
| |
| - | ====PCR purification====
| |
| - | {|class="wikitable"
| |
| - | !Sample number||Concentration(ng/µL)||Volume(µL)
| |
| - | |-
| |
| - | |E240①X-P||21.8||40
| |
| - | |-
| |
| - | |E240②X-P||32.4||45
| |
| - | |}
| |
| - | -20℃ freezer
| |
| - |
| |
| - | ====3. Error PCR====
| |
| - | {|class="wikitable"
| |
| - | !||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10µM)||Primer VR(10µM)||Template Δ1||Template||Template||KOD Pllus ver.2||Total
| |
| - | |-
| |
| - | |ΔTMD①||32||3||5||5||1.5||1.5||1||-||-||1||50
| |
| - | |-
| |
| - | |ΔTMD②||32||3||5||5||1.5||1.5||-||1||-||1||50
| |
| - | |-
| |
| - | |ΔTMD③||32||3||5||5||1.5||1.5||-||-||1||1||50
| |
| - | |}
| |
| - |
| |
| - | {|class="wikitable"
| |
| - | |94℃||2min||
| |
| - | |-
| |
| - | |98℃||10sec||rowspan="2"|20 cycles
| |
| - | |-
| |
| - | |68℃||4min
| |
| - | |-
| |
| - | |4℃||forever||
| |
| - | |}
| |
| - |
| |
| - | After the digestion by DpnI, we transfected 2µL of sample to 20µL of competent cell.
| |
| - |
| |
| - | ===Tuesday, August 3===
| |
| - |
| |
| - | ====1. The result of transformation====
| |
| - | {|class="wikitable"
| |
| - | |ΔTMD①||Many colonies
| |
| - | |-
| |
| - | |ΔTMD②||No colony
| |
| - | |-
| |
| - | |ΔTMD③||Many colonies
| |
| - | |}
| |
| - |
| |
| - | We picked two colonies from ΔTMD① and ΔTMD③, and cultured 37℃ 8/3~8/4.
| |
| - |
| |
| - | ====2. The construction of ML and MS====
| |
| - |
| |
| - | =====Miniprep=====
| |
| - | {|class="wikitable"
| |
| - | !Sample number||Concentration(ng/µL)
| |
| - | |-
| |
| - | |pSB4K5||60.7
| |
| - | |-
| |
| - | |LacP||26.8
| |
| - | |}
| |
| - |
| |
| - | =====RE=====
| |
| - | {|class="wikitable"
| |
| - | !||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
| |
| - | |-
| |
| - | |LacP||50||6||0.6||EcoRI||0.2||SpeI||0.2||3||60
| |
| - | |-
| |
| - | |pSB4K5(E-P)||50||6||0.6||EcoRI||0.2||PstI||0.2||3||60
| |
| - | |-
| |
| - | |E240①(X-P)||50||6||0.6||XbaI||0.2||PstI||0.2||3||60
| |
| - | |-
| |
| - | |E240②(X-P)||50||6||0.6||XbaI||0.2||PstI||0.2||3||60
| |
| - | |}
| |
| - | Incubate
| |
| - |
| |
| - | Purification
| |
| - |
| |
| - | =====PCR purification=====
| |
| - | {|class="wikitable"
| |
| - | !Sample number||Concentration(ng/µL)
| |
| - | |-
| |
| - | |pSB4K5 E-P||39.5
| |
| - | |-
| |
| - | |E240①X-P||21.8
| |
| - | |-
| |
| - | |E240②X-P||32.4
| |
| - | |}
| |
| - |
| |
| - | pSB4K5 E-P is concentrated 10µL and E240①X-P, E240②X-P are concentrated 1µL.
| |
| - |
| |
| - | Ethanol precipitation
| |
| - |
| |
| - | 1-5-G dilute milliQ 2µL
| |
| - |
| |
| - | =====Ligation=====
| |
| - | {|class="wikitable"
| |
| - | !||Vector||Insert 1||Insert 2||Ligation High||Total
| |
| - | |-
| |
| - | |ML1||pSB4K5 E-P 1||LacI E-S 1||E240①X-P 1||3||15
| |
| - | |-
| |
| - | |ML2||pSB4K5 E-P 1||LacI E-S 1||E240②X-P 1||3||15
| |
| - | |}
| |
| - | Incubation 17:30~20:20
| |
| - |
| |
| - | =====PCR of J23101-E240=====
| |
| - |
| |
| - | J23101-E240 is important in the measurement of RPU, so we amplified this parts by PCR.
| |
| - |
| |
| - | {|class="wikitable"
| |
| - | !||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10µM)||Primer VR(10µM)||Template J23101-E240||KOD plus ver.2 ||Total
| |
| - | |-
| |
| - | |MS①||32||3||5||5||1.5||1.5||1||1||50
| |
| - | |-
| |
| - | |MS②||32||3||5||5||1.5||1.5||-||1||50
| |
| - | |}
| |
| - |
| |
| - | {|class="wikitable"
| |
| - | |94℃||2min||
| |
| - | |-
| |
| - | |98℃||10sec||rowspan="3"|30 cycles
| |
| - | |-
| |
| - | |55℃||30sec
| |
| - | |-
| |
| - | |68℃||4min
| |
| - | |-
| |
| - | |4℃||forever||
| |
| - | |}
| |
| - |
| |
| - | =====PCR purification=====
| |
| - | {|class="wikitable"
| |
| - | !Sample number||Concentration(ng/µL)
| |
| - | |-
| |
| - | |J23101-E240||40.6
| |
| - | |}
| |
| - |
| |
| - | Discussion
| |
| - | J23101-E240(MS) is amplified corrrecly.
| |
| - |
| |
| - | =====RE=====
| |
| - | {|class="wikitable"
| |
| - | !||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
| |
| - | |-
| |
| - | |J23101-E240(E-P)||45||6||0.6||EcoRI||0.2||PstI||0.2||8||60
| |
| - | |}
| |
| - |
| |
| - | =====PCR purification=====
| |
| - | {|class="wikitable"
| |
| - | !Sample number||Concentration(ng/µL)||Volume(µL)
| |
| - | |-
| |
| - | |J23101-E240 E-P ||74.1||30
| |
| - | |}
| |
| - | J23101-E240 is concentrated 7µL
| |
| - |
| |
| - | =====Ligation=====
| |
| - | {|class="wikitable"
| |
| - | !||Vector||Insert||Ligation High||Total
| |
| - | |-
| |
| - | |MS||pSB4K5 E-P 1||J23101-E240 E-P 1||2||4
| |
| - | |}
| |
| - | Incubation 20:00~20:30
| |
| - |
| |
| - | =====Transformation=====
| |
| - | {|class="wikitable"
| |
| - | !Sample||Conc(/µL)||Sample Volume(µL)||Competent Cell(µL)||Total||Plate||Incubation
| |
| - | |-
| |
| - | |ML1||-||1||20||21||rowspan="3"|LB kan||rowspan="3"|8/3~8/4
| |
| - | |-
| |
| - | |ML2||-||1||20||21
| |
| - | |-
| |
| - | |MS||-||1||20||21
| |
| - | |}
| |
| - |
| |
| - | ===Thursday, August 5===
| |
| - |
| |
| - | ====1. Result of transformation====
| |
| - | {|class="wikitable"
| |
| - | |ML1||rowspan="3"|Many colonies
| |
| - | |-
| |
| - | |ML2
| |
| - | |-
| |
| - | |MS
| |
| - | |}
| |
| - |
| |
| - | ML1 and ML2
| |
| - |
| |
| - | pSB4K5 is inserted RFP generator. We didn't distinguish this inserted parts from low copy plasmid backbone, so self-ligated colony is red.
| |
| - | So, white colony is correctly inserted parts.
| |
| - | However, white colonies and green colonies are observed in ML1 and ML2 plate.
| |
| - | We cultured both white and green colonies.
| |
| - |
| |
| - | MS
| |
| - | Self-ligated colony is red Many of colonies are red, however, green colonies are observed.
| |
| - |
| |
| - | =====Culture and Master=====
| |
| - |
| |
| - | Green colony ML1-1 ML1-2 ML2-1 MS1-1 MS1-2 MS1-3
| |
| - | White colony ML1-3 ML1-4 ML2-2 ML2-3 ML2-4
| |
| - |
| |
| - | J23100 and LacP
| |
| - | 8/5~8/6
| |
| - |
| |
| - | ====2. Sequence====
| |
| - | {|class="wikitable"
| |
| - | !Sample number||Concentration(ng/µL)
| |
| - | |-
| |
| - | |ΔTMD①A||28.9
| |
| - | |-
| |
| - | |ΔTMD①B||25.3
| |
| - | |-
| |
| - | |ΔTMD③A||26.6
| |
| - | |-
| |
| - | |ΔTMD③B||24.0
| |
| - | |}
| |
| - |
| |
| - | As a result, deletion is succeeded, however, point mutation is failed. It is because DpnI is too little to digest all of template DNA.
| |
| - |
| |
| - | ===Friday, August 6===
| |
| - |
| |
| - | ====1. Miniprep====
| |
| - | ML1-1 ML1-2 ML1-3 ML1-4 ML2-1 ML2-2 ML2-3 ML2-4 MS1 MS2 MS3
| |
| - |
| |
| - | ====2. RE====
| |
| - | {|class="wikitable"
| |
| - | !Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
| |
| - | |-
| |
| - | |50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
| |
| - | |}
| |
| - |
| |
| - | =====Electrophoresis=====
| |
| - | 1 100bp 2λ 3λ 4100bp 5MS1 6MS2 7MS3 8ML1-1 9ML1-2 10ML1-3 11ML1-4 12ML2-1 13ML2-2 14ML2-3 15ML2-4 16MS1 17MS2
| |
| - |
| |
| - | 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
| |
| - |
| |
| - | [[image:KyotoExp100806-1.png]]
| |
| - |
| |
| - | Discussion
| |
| - | MS1 and MS2 are inserted correctly. ML1-1 and ML1-2 are inserted correctly. ML2-1 are inserted correctly.
| |
| - | White colonies are inserted not lacP but its vector. Top10 we used are deleted Lac operon.
| |
| - | Then, correctly inserted parts is green because of the lack of LacI.
| |
| - |
| |
| - | =====Error PCR(Retry)=====
| |
| - | {|class="wikitable"
| |
| - | !||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10µM)||Primer VR(10µM)||Template ΔTMD failed(50ng/µL)||KOD plus ver.2||Total
| |
| - | |-
| |
| - | |ΔTMD①||32||3||5||5||1.5||1.5||1||1||50
| |
| - | |-
| |
| - | |ΔTMD②||32||3||5||5||1.5||1.5||1||1||50
| |
| - | |}
| |
| - |
| |
| - | {|class="wikitable"
| |
| - | |94℃||2min
| |
| - | |-
| |
| - | |98℃||10sec||rowspan="2"|25 cycles
| |
| - | |-
| |
| - | |68℃||4min
| |
| - | |-
| |
| - | |4℃||forever||
| |
| - | |}
| |
| - |
| |
| - | Add DpnI 2µL and incubate 1h.
| |
| - |
| |
| - | =====Transformation=====
| |
| - | {|class="wikitable"
| |
| - | !Sample||Conc(/µL)||Sample Volum(µL)||Competent Cell(µL)||Total||Plate||Incubation
| |
| - | |-
| |
| - | |ΔTMD①||-||4||50||54||rowspan="3"|LB kan||rowspan="4"|8/6~8/9
| |
| - | |-
| |
| - | |ΔTMD②||-||4||50||54
| |
| - | |-
| |
| - | |2-17-F||-||2||50||52
| |
| - | |-
| |
| - | |2-I-5||||2||50||52||LB amp
| |
| - | |}
| |
"
