|
|
| (2 intermediate revisions not shown) |
| Line 1: |
Line 1: |
| - | {{:Team:Kyoto/Header}}
| |
| - | ==Notebook: Construction for Lysisbox==
| |
| - | ===Tuesday, July 20 <span class="by">By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto</span>===
| |
| - | ====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
| |
| - | {| class="experiments"
| |
| - | !Name||Well||Sample||Competent Cells||Total||Plate||Incubation||Result
| |
| - | |-
| |
| - | |<partinfo>J23100</partinfo>||1-18-C||1 µl||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 (Kan+)||×
| |
| - | |}
| |
| - | A vector of <partinfo>pSB4K5</partinfo> is Kanamycin-resistance, however, we plated it to LB plate (Amp+). And We didn't pre-culture <partinfo>B0015</partinfo> despite its vector is Kanamycin-resistance. So, it was predicted that we will fail the transformation of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo>.
| |
| | | | |
| - |
| |
| - | ===Wednesday, July 21 <span class="by">By: Wataru, Ken, Makoto, Takuya Y.</span>===
| |
| - | ====Culture at 37℃ from 07/21 20:50 to 07/22 17:00 and Making Master Plate====
| |
| - | ====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
| |
| - | {| class="experiments"
| |
| - | !Name||Well||Sample||Competent Cells||Total||Plate||Incubation||Result
| |
| - | |-
| |
| - | |<partinfo>pSB4K5</partinfo>||1-5-G||1 µl||20||21||rowspan="2"|LB (Kan+)||rowspan="2"|At 37℃, 7/21 20:50 - 7/22 16:30||○
| |
| - | |-
| |
| - | |<partinfo>B0015</partinfo>||1-23-L||1||20||21||○
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Stantard PCR|PCR]] for SRRz and S====
| |
| - | {| class="experiments"
| |
| - | !No.||Water||MgSO4||dNTPs||10xBuffer||Template DNA||Primer Fwd.||Primer Rev. (SRRz)||Primer Rev. (S)||KOD Plus ver.2||Total
| |
| - | |-
| |
| - | |1||28 µl||3||5||5||5||1.5||1.5||-||1||50
| |
| - | |-
| |
| - | |2||28||3||5||5||5||1.5||1.5||-||1||50
| |
| - | |-
| |
| - | |3||28||3||5||5||5||1.5||-||1.5||1||50
| |
| - | |-
| |
| - | |4||28||3||5||5||5||1.5||-||1.5||1||50
| |
| - | |-
| |
| - | |5||28||3||5||5||5||1.5||1.5||-||1||50
| |
| - | |-
| |
| - | |6||28||3||5||5||5||1.5||1.5||-||1||50
| |
| - | |-
| |
| - | |7||28||3||5||5||5||1.5||-||1.5||1||50
| |
| - | |-
| |
| - | |8||28||3||5||5||5||1.5||-||1.5||1||50
| |
| - | |}
| |
| - | {|class="experiments"
| |
| - | |-
| |
| - | |94℃||2min||
| |
| - | |-
| |
| - | |98℃||10sec||rowspan="3"|30 cycles
| |
| - | |-
| |
| - | |55℃||30sec
| |
| - | |-
| |
| - | |68℃||4min
| |
| - | |-
| |
| - | |4℃||forever||
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | ===Thursday, July 22 <span class="by">By: Wataru</span>===
| |
| - | ====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] (40min) of the PCR Products====
| |
| - | [[Image:KyotoExp100722-1.png|300px|right]]
| |
| - | {| class="electrophoresis"
| |
| - | !No.||Name||Length(bp)||Result
| |
| - | |-
| |
| - | |1||SRRz||1386||○
| |
| - | |-
| |
| - | |2||SRRz||1386||○
| |
| - | |-
| |
| - | |3||S||442||○
| |
| - | |-
| |
| - | |4||S||442||○
| |
| - | |-
| |
| - | |5||SRRz||1386||○
| |
| - | |-
| |
| - | |6||SRRz||1386||○
| |
| - | |-
| |
| - | |7||S||442||○
| |
| - | |-
| |
| - | |8||S||442||○
| |
| - | |}
| |
| - | Marker: 100bp, 1kb, 1kb, 100bp.
| |
| - | ====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
| |
| - | {| class="experiments"
| |
| - | !Name||Concentration
| |
| - | |-
| |
| - | |<partinfo>J23100</partinfo>||18.5 (ng/µl)
| |
| - | |-
| |
| - | |<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
| |
| - | |}
| |
| - | The concentration of all samples was very week. Probably our shaking incubation was week.
| |
| - | ====Culture from 07/22 17:00 to 07/23 10:00 and Making Master Plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo>====
| |
| - |
| |
| - |
| |
| - | ===Friday, July 23 <span class="by">By: Wataru, Tomo, Makoto</span>===
| |
| - | ====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
| |
| - | {| class="experiments"
| |
| - | !Name||Concentration
| |
| - | |-
| |
| - | |<partinfo>pSB4K5</partinfo>||79.2 (ng/µl)
| |
| - | |-
| |
| - | |<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.
| |
| - | ====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
| |
| - | {| class="experiments"
| |
| - | !No.||Name||Concentration||New Name
| |
| - | |-
| |
| - | |1||SRRz||18.6 ng/µl||-
| |
| - | |-
| |
| - | |3||S||77.6||S<sub>Sam7</sub>(1)
| |
| - | |-
| |
| - | |5||SRRz||33.6||-
| |
| - | |-
| |
| - | |7||S||65.4||S<sub>Sam7</sub>(2)
| |
| - | |}
| |
| - | The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.
| |
| - | ====[[Team:Kyoto/Protocols#Standard_PCR|Standard PCR]] for SRRz====
| |
| - | {| class="experiments"
| |
| - | !No.||Water||MgSO4||dNTPs||10xBuffer||Template DNA||Primer Fwd. (SRRz)||Primer Rev. (SRRz)||KOD plus ver.2||Total
| |
| - | |-
| |
| - | |1||28 µl||3||5||5||5||1.5||1.5||1||50
| |
| - | |-
| |
| - | |2||28||3||5||5||5||1.5||1.5||1||50
| |
| - | |-
| |
| - | |3||26.5||4.5||5||5||5||1.5||1.5||1||50
| |
| - | |-
| |
| - | |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
| |
| - | |}
| |
| - | {|class="experiments"
| |
| - | |-
| |
| - | |94℃||2min||
| |
| - | |-
| |
| - | |98℃||10sec||rowspan="3"|30 cycles
| |
| - | |-
| |
| - | |55℃||30sec
| |
| - | |-
| |
| - | |68℃||4min
| |
| - | |-
| |
| - | |4℃||forever||
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]] and [[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] (35min) to check function of our Restriction Enzyme====
| |
| - | {| class="experiments"
| |
| - | !No.||Name||Sample||10xBuffer||BSA||colspan="2"|Enzyme||MilliQ||Total||Incubation
| |
| - | |-
| |
| - | |1||<partinfo>J06702</partinfo>||5 µl||1||0.1||EcoRI||0.1||3.6||10||rowspan="5"|At 37℃ 7/23 18:00 - 7/23 18:30
| |
| - | |-
| |
| - | |2||<partinfo>J06702</partinfo>||5||1||0.1||XbaI||0.1||3.6||10
| |
| - | |-
| |
| - | |3||<partinfo>J06702</partinfo>||5||1||0.1||SpeI||0.1||3.6||10
| |
| - | |-
| |
| - | |4||<partinfo>J06702</partinfo>||5||1||0.1||PstI||0.1||3.6||10
| |
| - | |-
| |
| - | |5||<partinfo>J06702</partinfo>||5||1||0.1||colspan="2"|-||3.7||10
| |
| - | |}
| |
| - | [[Image:KyotoExp100723-1.png|300px|right]]
| |
| - | Marker: 1kb.
| |
| - | Comparison to No. 5 (control, circular DNA), the bands of No. 1, 2, 3, and 4 was shifted. The DNA of them was linearized by Restriction enzymes. So, our restriction enzymes work correctly.
| |
| - | ====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] and [[Team:Kyoto/Protocols#Ligation|Ligation]] to insert S gene to <partinfo>E0840</partinfo>====
| |
| - | {| class="experiments"
| |
| - | !Name||Sample||10xBuffer||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total||Incubation
| |
| - | |-
| |
| - | |S<sub>Sam7</sub>(1)||11 µl||5||EcoRI||0.2||SpeI||0.2||33.6||50||rowspan="3"|At 37℃ for 2h
| |
| - | |-
| |
| - | |S<sub>Sam7</sub>(2)||11||5||EcoRI||0.2||SpeI||0.2||33.6||50
| |
| - | |-
| |
| - | |<partinfo>E0840</partinfo>||45||5||EcoRI||0.2||XbaI||0.2||0||50
| |
| - | |}
| |
| - | After PCR Purification, evaporated them and diluted 3ul.
| |
| - | {| class="experiments"
| |
| - | !Name||colspan="2"|Vector||colspan="2"|Insert||Ligation High||Total
| |
| - | |-
| |
| - | |S<sub>Sam7</sub>(1)-E0840||<partinfo>E0840</partinfo>||0.5µl||S<sub>Sam7</sub>(1)||0.5||1||2
| |
| - | |-
| |
| - | |S<sub>Sam7</sub>(2)-E0840||<partinfo>E0840</partinfo>||0.5||S<sub>Sam7</sub>(2)||0.5||1||2
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | ===Monday, July 26 <span class="by">By: Wataru, Tomonori, Makoto</span>===
| |
| - | ====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of PCR Products====
| |
| - | [[Image:KyotoExp100726-1.png|300px|right]]
| |
| - | {| class="electrophoresis"
| |
| - | !No.||Name||Length(bp)||Result
| |
| - | |-
| |
| - | |1||SRRz||1386||
| |
| - | |-
| |
| - | |2||SRRz||1386||
| |
| - | |-
| |
| - | |3||SRRz||1386||
| |
| - | |-
| |
| - | |4||SRRz||1386||
| |
| - | |-
| |
| - | |5||SRRz||1386||
| |
| - | |-
| |
| - | |6||SRRz||1386||
| |
| - | |}
| |
| - | Marker: 1kb.
| |
| - | At the condition 4 (4.5µl MgSO4) and 6 (6µl MgSO4), SRRz is amplified very much. So we decided to use them.
| |
| - | ====PCR Purification====
| |
| - | {| class="experiments"
| |
| - | !No.||Name||Concentration||New Name
| |
| - | |-
| |
| - | |4||SRRZ||51.6 ng/µl||SRRz<sub>Sam7</sub>(1)
| |
| - | |-
| |
| - | |5||SRRZ||59.3||
| |
| - | |-
| |
| - | |6||SRRZ||59.6||SRRz<sub>Sam7</sub>(2)
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
| |
| - | {| class="experiments"
| |
| - | !Name||Well||Sample||Competent Cell||Total||Plate||Incubation||Result
| |
| - | |-
| |
| - | |<partinfo>E0240</partinfo>||1-12-M||1 µl||20||21||LB (Amp+)||rowspan="3"|At 37℃ 7/26 - 7/27||×
| |
| - | |-
| |
| - | |<partinfo>I20260</partinfo>||2-17-F||1||20||21||rowspan="2"|LB (Kan+)||×
| |
| - | |-
| |
| - | |<partinfo>J04450</partinfo>||1-5-E||1||20||21||×
| |
| - | |}
| |
| - | ====Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>====
| |
| - |
| |
| - |
| |
| - | ===Tuesday, July 27 <span class="by">By: Wataru, Tomo, Kazuya, Ken, Naoi===
| |
| - | ====[[Team:Kyoto/Protocols#Colony PCR|Colony PCR]] of S<sub>Sam7</sub>-E0840 (Electrophoresis for 35min)====
| |
| - | [[Image:KyotoExp100727-1.png|300px|right]]
| |
| - | {| class="electrophoresis"
| |
| - | !No.||Name||Length||Result
| |
| - | |-
| |
| - | |1||S<sub>Sam7</sub>(1)-E0840||1522||○
| |
| - | |-
| |
| - | |2||S<sub>Sam7</sub>(1)-E0840||1522||×
| |
| - | |-
| |
| - | |3||S<sub>Sam7</sub>(1)-E0840||1522||○
| |
| - | |-
| |
| - | |4||S<sub>Sam7</sub>(1)-E0840||1522||×
| |
| - | |-
| |
| - | |5||S<sub>Sam7</sub>(1)-E0840||1522||○
| |
| - | |-
| |
| - | |6||S<sub>Sam7</sub>(1)-E0840||1522||◎ (Use as S<sub>Sam7</sub>(1)-E0840)
| |
| - | |-
| |
| - | |7||S<sub>Sam7</sub>(2)-E0840||1522||×
| |
| - | |-
| |
| - | |8||S<sub>Sam7</sub>(2)-E0840||1522||×
| |
| - | |-
| |
| - | |9||S<sub>Sam7</sub>(2)-E0840||1522||×
| |
| - | |-
| |
| - | |10||S<sub>Sam7</sub>(2)-E0840||1522||×
| |
| - | |-
| |
| - | |11||S<sub>Sam7</sub>(2)-E0840||1522||◎ (Use as S<sub>Sam7</sub>(2)-E0840)
| |
| - | |-
| |
| - | |12||S<sub>Sam7</sub>(2)-E0840||1522||○
| |
| - | |-
| |
| - | |13||S<sub>Sam7</sub>(2)-E0840||1522||○
| |
| - | |-
| |
| - | | +||<partinfo>E0840</partinfo>||1116||
| |
| - | |-
| |
| - | | -||None||||
| |
| - | |}
| |
| - | Marker: 1kb, 100bp
| |
| - | ====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
| |
| - | {| class="experiments"
| |
| - | !Name||Concentration
| |
| - | |-
| |
| - | |<partinfo>R0011</partinfo>||26.9 ng/µl
| |
| - | |-
| |
| - | |<partinfo>B0015</partinfo>||120.0
| |
| - | |-
| |
| - | |<partinfo>E0840</partinfo>||120.1
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#RestrictionDigestion|Restriction Digestion]]====
| |
| - | {|class="experiments"
| |
| - | !||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total||Incubation
| |
| - | |-
| |
| - | |<partinfo>B0015</partinfo>||30 µl||5||0.5||EcoRI||0.4||XbaI||0.3||13.7||50||rowspan="3"|At 37℃ 16:45 - 18:00
| |
| - | |-
| |
| - | |SRRz<sub>Sam7</sub>(1)||40||5||0.5||EcoRI||0.4||SpeI||0.4||3.8||50
| |
| - | |-
| |
| - | |SRRz<sub>Sam7</sub>(2)||40||5||0.5||EcoRI||0.4||SpeI||0.4||3.8||50
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Ligation|Ligation]]====
| |
| - | ====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
| |
| - | {| class="experiments"
| |
| - | !Name||Sample||Competent Cells||Total||Plate||Incubation||Result
| |
| - | |-
| |
| - | |SRRz<sub>Sam7</sub>(1)-B0015|| || || ||rowspan="2"| ||rowspan="2"| ||○
| |
| - | |-
| |
| - | |SRRz<sub>Sam7</sub>(2)-B0015|| || || ||○
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | ===Wednesday, July 28 <span class="by">By: </span>===
| |
| - | ====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
| |
| - | {| class="experiments"
| |
| - | !Name||Concentration
| |
| - | |-
| |
| - | |S<sub>Sam7</sub>(1)-E0840||95.5 ng/µl
| |
| - | |-
| |
| - | |S<sub>Sam7</sub>(2)-E0840||98.6
| |
| - | |}
| |
| - | Diluted S<sub>Sam7</sub>(1)-E0840 and S<sub>Sam7</sub>(2)-E0840 20 times with water, and used as template DNA.
| |
| - | ====[[Team:Kyoto/Protocols#Mutagenesis (Point mutation, Deletion, Insertion)|Deletion PCR]] to delete a functional domain of S gene====
| |
| - | {| class="experiments"
| |
| - | !||Water||MgSO4||dNTPs||10xBuffer||Primer Fwd.||Primer Rev.||S<sub>Sam7</sub>(1)-E0840||S<sub>Sam7</sub>(2)-E0840||KOD Plus ver.2||Total
| |
| - | |-
| |
| - | |S<sub>Sam7,ΔTMD1</sub>(1)-E0840 (1)||28||3||5||5||1.5||1.5||5||-||1||50
| |
| - | |-
| |
| - | |S<sub>Sam7,ΔTMD1</sub>(1)-E0840 (2)||28||3||5||5||1.5||1.5||5||-||1||50
| |
| - | |-
| |
| - | |S<sub>Sam7,ΔTMD1</sub>(2)-E0840 (1)||28||3||5||5||1.5||1.5||-||5||1||50
| |
| - | |-
| |
| - | |S<sub>Sam7,ΔTMD1</sub>(2)-E0840 (2)||28||3||5||5||1.5||1.5||-||5||1||50
| |
| - | |}
| |
| - | {| class="experiments"
| |
| - | |94℃||2min||
| |
| - | |-
| |
| - | |98℃||10sec||rowspan="3"|35 cycles
| |
| - | |-
| |
| - | |55℃||30sec
| |
| - | |-
| |
| - | |68℃||4min
| |
| - | |-
| |
| - | |4℃||forever||
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] to check the function of DpnI====
| |
| - | {| class="experiments"
| |
| - | !Name||Sample||fast digestion buffer||DpnI||MilliQ||Total
| |
| - | |-
| |
| - | |S<sub>Sam7,ΔTMD1</sub>(1)-E0840 (1)||3||1||0.1||5.8||10
| |
| - | |-
| |
| - | |S<sub>Sam7,ΔTMD1</sub>(2)-E0840 (2)||3||1||0.1||5.8||10
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] for 35min====
| |
| - | [[image:KyotoExp100728-1.png|300px|right]]
| |
| - | {| class="electrophoresis"
| |
| - | !No.||Name||Length||Result
| |
| - | |-
| |
| - | |1||Not digested S<sub>Sam7</sub>(1)-E0840||3363||
| |
| - | |-
| |
| - | |2||Not digested S<sub>Sam7</sub>(2)-E0840||3363||
| |
| - | |-
| |
| - | |3||Digested S<sub>Sam7</sub>(1)-E0840||1021, 933, 402, 341, 258, 105, ...||
| |
| - | |-
| |
| - | |4||Digested S<sub>Sam7</sub>(2)-E0840||1021, 933, 402, 341, 258, 105, ...||
| |
| - | |}
| |
| - | Marker: 1kb, 100bp
| |
| - | DpnI works correctly.
| |
| - |
| |
| - |
| |
| - | ===Thursday, July 29 <span class="by">By: </span>===
| |
| - | ====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]]====
| |
| - | {| class="experiments"
| |
| - | !Name||Sample volume||Fastdigestion Buffer||colspan="2"|Enzyme 1||MilliQ||Total||Incubation
| |
| - | |-
| |
| - | |S<sub>Sam7,ΔTMD1</sub>(1)-E0840 (1)||50 µl||6||DpnI||0.2||3.8||60||rowspan="2"|07/29 09:40 - 07/29 11:00
| |
| - | |-
| |
| - | |S<sub>Sam7,ΔTMD1</sub>(2)-E0840 (1)||50||6||DpnI||0.2||3.8||60
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Ligation|Ligation]] and [[Team:Kyoto/Protocols#Phosphorylation|Phosphorylation]]====
| |
| - | {| class="experiments"
| |
| - | !Name||Sample||MilliQ||Ligation High||T4 Kinase||Total||Incubation
| |
| - | |-
| |
| - | |S<sub>Sam7,ΔTMD1</sub>(1)-E0840 (1)||2 µl||7||5||1||15||rowspan="2"|07/29 11:30 ~ 07/29 13:00
| |
| - | |-
| |
| - | |S<sub>Sam7,ΔTMD1</sub>(1)-E0840 (2)||2||7||5||1||15
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
| |
| - | {| class="experiments"
| |
| - | !Name||Sample Volume||Competent Cell||Total||Plate||Incubation||Result
| |
| - | |-
| |
| - | |S<sub>Sam7,ΔTMD1</sub>(1)-E0840 (1)||3 µl||30||33||rowspan="2"|LB Amp+||rowspan="2"|07/29 ~ 07/30||○
| |
| - | |-
| |
| - | |S<sub>Sam7,ΔTMD1</sub>(1)-E0840 (2)||3||30||33||○
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | ===Monday, August 2 <span class="by">By: Wataru, Ken</span>===
| |
| - | ====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
| |
| - | {|class="experiments"
| |
| - | !Name||Concentration(ng/µL)
| |
| - | |-
| |
| - | |S<sub>ΔTMD1</sub>-E0840-1||52.7
| |
| - | |-
| |
| - | |S<sub>ΔTMD1</sub>-E0840-2||54.4
| |
| - | |-
| |
| - | |S<sub>ΔTMD1</sub>-E0840-3||89.5
| |
| - | |-
| |
| - | |<partinfo>pSB4K5</partinfo>||50.7
| |
| - | |-
| |
| - | |<partinfo>R0011</partinfo>||18.6
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Standard_PCR|Standard PCR]] of <partinfo>E0240</partinfo>====
| |
| - | 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="experiments"
| |
| - | !Name||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10µM)||Primer VR(10µM)||Template E240||KOD Pllus ver.2||Total
| |
| - | |-
| |
| - | |E0240<sub>1</sub>||28||3||5||5||1.5||1.5||5||1||50
| |
| - | |-
| |
| - | |E0240<sub>2</sub>||28||3||5||5||1.5||1.5||5||1||50
| |
| - | |}
| |
| - | {|class="experiments"
| |
| - | |94℃||2min||
| |
| - | |-
| |
| - | |98℃||10sec||rowspan="3"|35 cycles
| |
| - | |-
| |
| - | |55℃||30sec
| |
| - | |-
| |
| - | |68℃||4min
| |
| - | |-
| |
| - | |4℃||forever||
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]]====
| |
| - | ====PCR Purification====
| |
| - | {|class="experiments"
| |
| - | !Sample number||Concentration(ng/µL)
| |
| - | |-
| |
| - | |E0240<sub>1</sub>||42.6
| |
| - | |-
| |
| - | |E0240<sub>2</sub>||55.3
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]] for inserting <partinfo>E0240</partinfo> to pSB4K5 by 3A assembly====
| |
| - | {| class="experiments"
| |
| - | !Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
| |
| - | |-
| |
| - | |E0240<sub>1</sub>(X-P)||30||5||0.5||XbaI||0.2||PstI||0.2||14.1||50
| |
| - | |-
| |
| - | |E0240<sub>2</sub>(X-P)||30||5||0.5||XbaI||0.2||PstI||0.2||14.1||50
| |
| - | |}
| |
| - | ====PCR Purification====
| |
| - | {| class="experiments"
| |
| - | !Name||Concentration(ng/µL)||Volume(µL)
| |
| - | |-
| |
| - | |E0240<sub>1</sub>(X-P)||21.8||40
| |
| - | |-
| |
| - | |E0240<sub>2</sub>(X-P)||32.4||45
| |
| - | |}
| |
| - | Stored at -20℃.
| |
| - | ====Error PCR====
| |
| - | {|class="experiments"
| |
| - | !Name||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
| |
| - | |-
| |
| - | |S<sub>ΔTMD1</sub>-E0840<sub>1</sub>-1||32||3||5||5||1.5||1.5||1||-||-||1||50
| |
| - | |-
| |
| - | |S<sub>ΔTMD1</sub>-E0840<sub>1</sub>-2||32||3||5||5||1.5||1.5||-||1||-||1||50
| |
| - | |-
| |
| - | |S<sub>ΔTMD1</sub>-E0840<sub>2</sub>||32||3||5||5||1.5||1.5||-||-||1||1||50
| |
| - | |}
| |
| - | {|class="experiments"
| |
| - | |94℃||2min||
| |
| - | |-
| |
| - | |98℃||10sec||rowspan="2"|20 cycles
| |
| - | |-
| |
| - | |68℃||4min
| |
| - | |-
| |
| - | |4℃||forever||
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
| |
| - | {| class="experiments"
| |
| - | !Name||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
| |
| - | |-
| |
| - | |S<sub>ΔTMD1</sub>-E0840<sub>1</sub>-1||2||20||22||rowspan="3"|||rowspan="3"|||○
| |
| - | |-
| |
| - | |S<sub>ΔTMD1</sub>-E0840<sub>1</sub>-2||2||20||22||}
| |
| - | |-
| |
| - | |S<sub>ΔTMD1</sub>-E0840<sub>2</sub>||2||20||22||○
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | ===Tuesday, August 3 <span class="by">By: </span>===
| |
| - | ====Culture of each two colonies of S<sub>ΔTMD1</sub>-E0840<sub>1</sub>-1 and S<sub>ΔTMD1</sub>-E0840<sub>2</sub> for 37℃ 08/03-08/04====
| |
| - | ====[[Team:Kyoto/Protocols#Miniprep|Miniprep]] for Construction of Measure(''lac''P) and Measure(Standard)====
| |
| - | {|class="experiments"
| |
| - | !Sample number||Concentration(ng/µL)
| |
| - | |-
| |
| - | |<partinfo>pSB4K5</partinfo>||60.7
| |
| - | |-
| |
| - | |<partinfo>R0011</partinfo>||26.8
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]]====
| |
| - | {|class="experiments"
| |
| - | !Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
| |
| - | |-
| |
| - | |R0011||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
| |
| - | |-
| |
| - | |E0240<sub>1</sub>(X-P)||50||6||0.6||XbaI||0.2||PstI||0.2||3||60
| |
| - | |-
| |
| - | |E0240<sub>2</sub>(X-P)||50||6||0.6||XbaI||0.2||PstI||0.2||3||60
| |
| - | |}
| |
| - | ====PCR Purification====
| |
| - | {|class="experiments"
| |
| - | !Sample number||Concentration(ng/µL)
| |
| - | |-
| |
| - | |pSB4K5(E-P)||39.5
| |
| - | |-
| |
| - | |E0240<sub>1</sub>(X-P)||21.8
| |
| - | |-
| |
| - | |E0240<sub>2</sub>(X-P)||32.4
| |
| - | |}
| |
| - | pSB4K5(E-P) is concentrated 10µL and E0240<sub>1</sub>(X-P), E0240<sub>2</sub>(X-P) are concentrated 1µL.
| |
| - | ====Ethanol Precipitation====
| |
| - | ====Dilution of <partinfo>pSB4K5</partinfo> by 2µl MilliQ====
| |
| - | ====[[Team:Kyoto/Protocols#Ligation|Ligation]]====
| |
| - | {|class="experiments"
| |
| - | !||Vector||colspan="2"|Insert 1||colspan="2"|Insert 2||Ligation High||Total||Incubation
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]||pSB4K5(E-P)||1||R0011(E-S)||1||E0240<sub>1</sub>(X-P)||1||3||15||rowspan="2"|17:30 - 20:20
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]||pSB4K5(E-P)||1||R0011(E-S)||1||E0240<sub>2</sub>(X-P)||1||3||15
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Standard PCR|Standard PCR]] of <partinfo>J23101</partinfo>-<partinfo>E0240</partinfo> that is important in the measurement of RPU====
| |
| - | {|class="experiments"
| |
| - | !Name||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10µM)||Primer VR(10µM)||Template J23101-E0240||KOD plus ver.2 ||Total
| |
| - | |-
| |
| - | |J23101-E0240<sub>1</sub>||32||3||5||5||1.5||1.5||1||1||50
| |
| - | |-
| |
| - | |J23101-E0240<sub>2</sub>||32||3||5||5||1.5||1.5||-||1||50
| |
| - | |}
| |
| - | {|class="experiments"
| |
| - | |94℃||2min||
| |
| - | |-
| |
| - | |98℃||10sec||rowspan="3"|30 cycles
| |
| - | |-
| |
| - | |55℃||30sec
| |
| - | |-
| |
| - | |68℃||4min
| |
| - | |-
| |
| - | |4℃||forever||
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
| |
| - | {|class="experiments"
| |
| - | !Name||Concentration(ng/µL)
| |
| - | |-
| |
| - | |J23101-E0240||40.6
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]====
| |
| - | {|class="experiments"
| |
| - | !Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
| |
| - | |-
| |
| - | |J23101-E0240(E-P)||45||6||0.6||EcoRI||0.2||PstI||0.2||8||60
| |
| - | |}
| |
| - | ====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
| |
| - | {| class="experiments"
| |
| - | !Name||Concentration(ng/µL)||Volume(µL)
| |
| - | |-
| |
| - | |J23101-E0240(E-P)||74.1||30
| |
| - | |}
| |
| - | J23101-E0240(E-P) is concentrated 7µL
| |
| - | ====[[Team:Kyoto/Protocols#Ligation|Ligation]]====
| |
| - | {|class="experiments"
| |
| - | !||Vector||colspan="2"|Insert||colspan="2"|Ligation High||Total||Incubation
| |
| - | |-
| |
| - | |J23101-E0240[Low]||pSB4K5(E-P)||1||J23101-E0240(E-P)||1||2||4||20:00-20:30
| |
| - | |}
| |
| - | <div class="measure-construction"><br />
| |
| - | ====Transformation====
| |
| - | {| class="experiments"
| |
| - | !Name||Conc(/µL)||Sample Volume(µL)||Competent Cell(µL)||Total||Plate||Incubation
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]||-||1||20||21||rowspan="3"|LB kan||rowspan="3"|8/3~8/4
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]||-||1||20||21
| |
| - | |-
| |
| - | |J23101-E0240[Low]||-||1||20||21
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | ===Thursday, August 5 <span class="by">By: </span>===
| |
| - | ====Result of Transformation====
| |
| - | {|class="experiments"
| |
| - | |R0011-E0240<sub>1</sub>[Low]||rowspan="3"|Many colonies
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]
| |
| - | |-
| |
| - | |J23101-E0240[Low]
| |
| - | |}
| |
| - | 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 R0011-E0240<sub>1</sub>[Low] and R0011-E0240<sub>2</sub>[Low] plate. We cultured both white and green colonies.
| |
| - | In J23101-E0240[Low], Many of colonies are red, but green colonies are observed. We cultured green colonies.
| |
| - | {| class="experiments"
| |
| - | !Name||Color||Incubation
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]-1||Green Colony||rowspan="11"|8/5-8/6
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]-2||Green Colony
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]-3||White Colony
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]-4||White Colony
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]-1||Green Colony
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]-2||White Colony
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]-3||White Colony
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]-4||White Colony
| |
| - | |-
| |
| - | |J23101-E0240[Low]-1||Green Colony
| |
| - | |-
| |
| - | |J23101-E0240[Low]-2||Green Colony
| |
| - | |-
| |
| - | |J23101-E0240[Low]-3||Green Colony
| |
| - | |}
| |
| - | {| class="experiments"
| |
| - | !Name||Concentration(ng/µL)
| |
| - | |-
| |
| - | |S<sub>ΔTMD1</sub>-E0840<sub>1</sub>-1-A||28.9
| |
| - | |-
| |
| - | |S<sub>ΔTMD1</sub>-E0840<sub>1</sub>-1-B||25.3
| |
| - | |-
| |
| - | |S<sub>ΔTMD1</sub>-E0840<sub>2</sub>-A||26.6
| |
| - | |-
| |
| - | |S<sub>ΔTMD1</sub>-E0840<sub>2</sub>-B||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===
| |
| - | ====Miniprep====
| |
| - | {| class="experiments"
| |
| - | !Name
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]-1
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]-2
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]-3
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]-4
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]-1
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]-2
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]-3
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]-4
| |
| - | |-
| |
| - | |J23101-E0240[Low]-1
| |
| - | |-
| |
| - | |J23101-E0240[Low]-2
| |
| - | |-
| |
| - | |J23101-E0240[Low]-3
| |
| - | |}
| |
| - | ====Restriction Digestion====
| |
| - | {|class="experiments"
| |
| - | !Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]-1||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]-2||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]-3||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
| |
| - | |-
| |
| - | |R0011-E0240<sub>1</sub>[Low]-4||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]-1||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]-2||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]-3||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
| |
| - | |-
| |
| - | |R0011-E0240<sub>2</sub>[Low]-4||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
| |
| - | |-
| |
| - | |J23101-E0240[Low]-1||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
| |
| - | |-
| |
| - | |J23101-E0240[Low]-2||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
| |
| - | |-
| |
| - | |J23101-E0240[Low]-3||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
| |
| - | |}
| |
| - | =====Electrophoresis=====
| |
| - | {| class="experiments"
| |
| - | |M||100bp||colspan="4"|||colspan="4"|
| |
| - | |-
| |
| - | |M||λ
| |
| - | |-
| |
| - | |M||λ
| |
| - | |-
| |
| - | |M||100bp
| |
| - | |-
| |
| - | |1||J23101-E0240[Low]-1||||
| |
| - | |-
| |
| - | |2||J23101-E0240[Low]-2||||
| |
| - | |-
| |
| - | |3||J23101-E0240[Low]-1||||
| |
| - | |-
| |
| - | |4||R0011-E0240<sub>1</sub>[Low]-1||||○
| |
| - | |-
| |
| - | |5||R0011-E0240<sub>1</sub>[Low]-2||||○
| |
| - | |-
| |
| - | |6||R0011-E0240<sub>1</sub>[Low]-3||||}
| |
| - | |-
| |
| - | |7||R0011-E0240<sub>1</sub>[Low]-4||||}
| |
| - | |-
| |
| - | |8||R0011-E0240<sub>2</sub>[Low]-1||||○
| |
| - | |-
| |
| - | |9||R0011-E0240<sub>2</sub>[Low]-2||||}
| |
| - | |-
| |
| - | |10||R0011-E0240<sub>2</sub>[Low]-3||||}
| |
| - | |-
| |
| - | |11||R0011-E0240<sub>2</sub>[Low]-4||||}
| |
| - | |-
| |
| - | |12||J23101-E0240[Low]-1||||○
| |
| - | |-
| |
| - | |13||J23101-E0240[Low]-2||||○
| |
| - | |}
| |
| - | [[Image:KyotoExp100806-1.png]]
| |
| - | White colonies are not inserted <partinfo>R0011</partinfo> but its vector. Top10 we used are deleted Lac operon. Then, correctly inserted parts is green because of the lack of ''lacI'' gene.
| |
| - | ====Error PCR (Retry)====
| |
| - | {| class="experiments"
| |
| - | !Name||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="experiments"
| |
| - | |94℃||2min
| |
| - | |-
| |
| - | |98℃||10sec||rowspan="2"|25 cycles
| |
| - | |-
| |
| - | |68℃||4min
| |
| - | |-
| |
| - | |colspan="2"|Add DpnI 2µl
| |
| - | |-
| |
| - | |Incubate||1h
| |
| - | |-
| |
| - | |4℃||forever||
| |
| - | |}
| |
| - | ====Transformation====
| |
| - | {| class="experiments"
| |
| - | !Name||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
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | ===Monday, August 9 <span class="by">By: Wataru, Tomonori, Ken, Takuya</span>===
| |
| - | ====Miniprep of MS and ML====
| |
| - | {|class="experiments"
| |
| - | !Sample number||concentration(ng/µL)
| |
| - | |-
| |
| - | |MS||116.2
| |
| - | |-
| |
| - | |ML||146.6
| |
| - | |}
| |
| - | ====Transfotrmation of MS and ML====
| |
| - | {|class="experiments"
| |
| - | !Sample||conc(ng/µL)||Sample vol(µL)||Competent Cell||Competent cell vol(µL)||Total vol(µL)||Plate||Incuvation
| |
| - | |-
| |
| - | |MS||116.2||2||KRX||50||52||rowspan="2"|LB kanamycin||rowspan="2"|8/9 18:00‾8/10 12:00
| |
| - | |-
| |
| - | |ML||146.6||2||KRX||50||52
| |
| - | |}
| |
| - | ====Restriction enzyme digestion and ethanol precipitation====
| |
| - | To use lac p for next ligation, we digested 1-6-G by EroRI and PstI
| |
| - | {|class="experiments"
| |
| - | !Sample||10x Buffer||BSA||Enzyme (EcoRI)||Enzyme (PstI)||MilliQ||Total
| |
| - | |-
| |
| - | |50||6||0.6||0.5||0.5||2.4||60
| |
| - | |}
| |
| - | Incubate 37℃ 8/9 16:20‾18:20
| |
| - | After restriction enzyme digestion, we did ethanol precipitation.
| |
| - | ====Ligation and Transformation====
| |
| - | {|class="experiments"
| |
| - | !Sample||Conc (nu/µL)||Sample vol (µL)||Competent cell||Competent cell vol (µL)||Total vol (µL)||Plate||Incuvation
| |
| - | |-
| |
| - | |rowspan="2"|Lac p (low)||rowspan="2"|-||2||KRX||50||52||rowspan="2"|LB kanamycin||rowspan="2"|8/9 20:00‾8/10 9:00
| |
| - | |-
| |
| - | |2||C2||50||52
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | ===Tuesday, August 10 <span class="by">By: Wataru, Tomonori, Ken, Fumitaka</span>
| |
| - | ====Making culture plate on lac p (low), MS and ML====
| |
| - | {|class="experiments"
| |
| - | |rowspan="2"|Lac p (low)||KRX||rowspan="4"|Many colonies
| |
| - | |-
| |
| - | |C2
| |
| - | |-
| |
| - | |MS||KRX
| |
| - | |-
| |
| - | |ML||KRX
| |
| - | |}
| |
| - | ====Minprep of ΔTMD1+GFP====
| |
| - | {|class="experiments"
| |
| - | !Sample number||Concentration (ng/µL)
| |
| - | |-
| |
| - | |1-1||9.9
| |
| - | |-
| |
| - | |1-2||27.3
| |
| - | |-
| |
| - | |2-1||43.2
| |
| - | |-
| |
| - | |2-2||34.7
| |
| - | |}
| |
| - | 37℃ 8/9 18:00‾8/10 9:00
| |
| - | ====Culture and Master Plate====
| |
| - |
| |
| - |
| |
| - | ===Wednesday, August 11 <span class="by">By: Wataru, Naoi, Ken, Takuya</span>
| |
| - | {| class="experiments"
| |
| - | !Sample||Medium||Cloud||Incubation
| |
| - | |-
| |
| - | |rowspan="2"|1||Kanamycin||o||rowspan="14"|37℃8/10 20:00‾8/11 9:00
| |
| - | |-
| |
| - | |Ampicillin||x
| |
| - | |-
| |
| - | |rowspan="2"|2||Kanamycin||o
| |
| - | |-
| |
| - | |Ampicillin||o
| |
| - | |-
| |
| - | |rowspan="2"|3||Kanamycin||o
| |
| - | |-
| |
| - | |Ampicillin||x
| |
| - | |-
| |
| - | |rowspan="2"|4||Kanamycin||o
| |
| - | |-
| |
| - | |Ampicillin||x
| |
| - | |-
| |
| - | |rowspan="2"|5||Kanamycin||o
| |
| - | |-
| |
| - | |Ampicillin||x
| |
| - | |-
| |
| - | |rowspan="2"|6||Kanamycin||o
| |
| - | |-
| |
| - | |Ampicillin||o
| |
| - | |-
| |
| - | |rowspan="2"|7||Kanamycin||o
| |
| - | |-
| |
| - | |Ampicillin||x
| |
| - | |}
| |
| - | Discussion: About sample 1, 3, 4, 5 and 7, lac promoter was correctly inserted in low copy plasmid. About sample 2 and 6, low copy plasmid and vector derived from lac promoter were ligated. We decided to use sample 1 or 3.
| |
| - | ====Miniprep of C2+lac(low), S-R-Rz 1', 3'====
| |
| - | lac(low)1 : 31.2 (ng/µL)
| |
| - | lac(low)2 : 29.9 (ng/µL)
| |
| - | ====Restriction Digestion and electrophoresis of lac (low) 1 and 3====
| |
| - | {| class="experiments"
| |
| - | !Name||EcoRI||PstI
| |
| - | |-
| |
| - | |1||0.2||-
| |
| - | |-
| |
| - | |2||-||0.2
| |
| - | |-
| |
| - | |3||0.2||0.2
| |
| - | |-
| |
| - | |N||-||-
| |
| - | |}
| |
| - | Sample: 1-1, 1-2, 1-3, 1-N, 3-1, 3-2, 3-3, 3-N
| |
| - | M 1-1 1-2 1-3 1-N M M 3-1 3-2 3-3 3-N M
| |
| - | [[image:KyotoExp100811-1.png]]
| |
| - | Discussion: Each enzyme correctly cut samples.
| |
| - | ====Screening PCR of SRRz====
| |
| - | Sample: 1-20
| |
| - | Control: P(1-23L) P'(2-8E) N
| |
| - | Maker: lambda
| |
| - | M N P P' P 1 2 3 4 5 6 M
| |
| - | [[image:KyotoExp100811-2.png]]
| |
| - | 7 8 9 10 11 12 13 M 14 15 16 18 19 20 M
| |
| - | [[image:KyotoExp100811-3.png]]
| |
| - | Discussion: All of the sample were self-ligation of DT and SRRz weren't inserted.
| |
| - |
| |
| - |
| |
| - | ===Thursday, August 12 <span class="by">By: Wataru, Ken</span>===
| |
| - | ====Restriction Digestion and electrophoresis of <partinfo>B0015</partinfo>====
| |
| - | {| class="experiments"
| |
| - | !Sample name||Template||10xbuffer||100xbuffer||EcoRI||XbaI 1||XbaI 2||SpeI||PstI 1||PstI 2||Water||Total
| |
| - | |-
| |
| - | |1||3||1||0.1||0.2||-||-||-||-||-||5.7||10
| |
| - | |-
| |
| - | |2||3||1||0.1||-||0.2||-||-||-||-||5.7||10
| |
| - | |-
| |
| - | |3||3||1||0.1||-||-||0.2||-||-||-||5.7||10
| |
| - | |-
| |
| - | |4||3||1||0.1||-||-||-||0.2||-||-||5.7||10
| |
| - | |-
| |
| - | |5||3||1||0.1||-||-||-||-||0.2||-||5.7||10
| |
| - | |-
| |
| - | |6||3||1||0.1||-||-||-||-||-||0.2||5.7||10
| |
| - | |-
| |
| - | |N||3||1||0.1||-||-||-||-||-||-||5.9||10
| |
| - | |}
| |
| - | Sample: 1-6, N Maker: lambda, 100
| |
| - | M 1 2 3 4 5 6 N M M M
| |
| - | [[image:KyotoExp100812-1.png]]
| |
| - | Discussion: Each enzyme correctly cut each sample and was active.
| |
| - |
| |
| - |
| |
| - | ===Thursday, August 19 <span class="by">By: Wataru, Tomo, Ken</span>
| |
| - | ====Miniprep of SΔTMD1GFP====
| |
| - | 29.6(ng/µg)
| |
| - | ====Point mutation PCR of ΔTMD1GFP====
| |
| - | {| class="experiments"
| |
| - | !Sample number||Template||10xbuffer||dNTPs||MgSO4||Primer 1||Primer 2||Water||KOD-plus-||Total
| |
| - | |-
| |
| - | |1||1.5||5||5||3||1.5||1.5||31.5||1||50
| |
| - | |-
| |
| - | ||2||1.5||5||5||3||1.5||1.5||31.5||1||50
| |
| - | |-
| |
| - | |control||1.5||5||5||3||1.5||1.5||32.5||-||50
| |
| - | |}
| |
| - | {| class="experiments"
| |
| - | |94(℃)||2min||
| |
| - | |-
| |
| - | |98||10sec||rowspan="3"|30cycles
| |
| - | |-
| |
| - | |55||30sec
| |
| - | |-
| |
| - | |68||3.5min
| |
| - | |-
| |
| - | |4.0||forever||
| |
| - | |}
| |
| - | ====Restriction Digestion(DpnI): 17:50-18:50====
| |
| - | ====Electrophoresis====
| |
| - | Sample: 1, 2, Control Marker: lambda, 100
| |
| - | [[Image:KyotoExp100819-1.png]]
| |
| - | ====Ligation and Transformation====
| |
| - | We named point mutation PCR products rΔTMD1GFP.
| |
| - |
| |
| - |
| |
| - | ===Monday, August 23 <span class="by">By: Wataru, Tomo, Ken, Fumitaka, Tasuku</span>===
| |
| - | ====Miniprep of ΔTMD1====
| |
| - | {| class="experiments"
| |
| - | |Sample number||Concentration(ng/µg)
| |
| - | |-
| |
| - | |1-1||58.9
| |
| - | |-
| |
| - | |2-2||49.9
| |
| - | |}
| |
| - | ====Sequencing of ΔTMD1 and MS====
| |
| - | Sample: rδTMD1GFP1-1, 2-2, and MS
| |
| - | Discussion: The sequencing was in success and the results were desirable. It meant point mutation of δTMD1GFP was succeeded and sequence of MS was confirmed. We decided to use rδTMD1GFP.
| |
| - | ====Screening PCR of SRRz-DT====
| |
| - | Sample: 1-13, Marker: lambda and 100, Control:P(1-23L) and N
| |
| - | {| class="experiments"
| |
| - | |90℃||10min||
| |
| - | |-
| |
| - | |94℃||30sec||rowspan="3"|35cycles
| |
| - | |-
| |
| - | |50℃||30sec
| |
| - | |-
| |
| - | |72℃||1.5min
| |
| - | |-
| |
| - | |72℃||4min||
| |
| - | |-
| |
| - | |4℃||hold||
| |
| - | |}
| |
| - | M 1 2 3 4 5 6 7 8 9 10 11 12 13 P N M
| |
| - | [[Image:KyotoExp100823-1.png]]
| |
| - | Discussion: We found the band; about 200bp, and it meant the lligation was completed successfully.
| |
| - | ====Deletion PCR of rΔTMD1GFP 2-2====
| |
| - | {| class="experiments"
| |
| - | !Sample||10x||dNTPs||Primer1||Primer2||Template||Water||KOD-plus-||Total
| |
| - | |-
| |
| - | |1||5||5||1.5||1.5||1||35||1||50
| |
| - | |-
| |
| - | |2||5||5||1.5||1.5||1||35||1||50
| |
| - | |-
| |
| - | |Control||5||5||1.5||1.5||1||35||-||50
| |
| - | |}
| |
| - | {| class="experiments"
| |
| - | |94℃||2min||
| |
| - | |-
| |
| - | |94℃||10sec||rowspan="3"|35cycles
| |
| - | |-
| |
| - | |56℃||30sec
| |
| - | |-
| |
| - | |68℃||3.5min
| |
| - | |-
| |
| - | |4℃||hold||
| |
| - | |}
| |
| - | ====Restriction Digestion(DpnI)====
| |
| - | {| class="experiments"
| |
| - | |Template||25(µL)
| |
| - | |-
| |
| - | |DpnI||1
| |
| - | |-
| |
| - | |Total||26
| |
| - | |}
| |
| - | 19:10-20:10
| |
| - | ====Ligation====
| |
| - | {|class="experiments"
| |
| - | !Sample||Template||Water||Ligation high||T4 Kinase||total
| |
| - | |-
| |
| - | |1||3||6||5||1||15
| |
| - | |-
| |
| - | |2||3||6||5||1||15
| |
| - | |-
| |
| - | |Control||3||6||5||1||15
| |
| - | |}
| |
| - | 20:15-21:15
| |
| - | ====Transformation====
| |
| - | We named sample 1, 2 and control rrδTMD1GFP1, 2 and control.
| |
| - |
| |
| - |
| |
| - | ===Tuesday, August 24 <span class="by">By:Ken, Tomo, Tasuku, Takuya</span>===
| |
| - | ====Retry of deletion PCR of rδTMD1 GFP====
| |
| - | {| class="experiments"
| |
| - | !Sample||10x||dNTPs||MgSO4||Primer1||Primer2||Template||Water||KOD-plus-||Total
| |
| - | |-
| |
| - | |1||5||5||3||1.5||1.5||1||32||1||50
| |
| - | |-
| |
| - | |2||5||5||3||1.5||1.5||1||32||1||50
| |
| - | |-
| |
| - | |Control||5||5||3||1.5||1.5||1||32||1||50
| |
| - | |}
| |
| - | {|class="experiments"
| |
| - | |94℃||2min||
| |
| - | |-
| |
| - | |94℃||10sec||rowspan="3"|35cycles
| |
| - | |-
| |
| - | |58℃||30sec
| |
| - | |-
| |
| - | |68℃||3.5min
| |
| - | |-
| |
| - | |4℃||hold||
| |
| - | |}
| |
| - | ====Restriction Digestion (DpnI)====
| |
| - | 14:15-15:15
| |
| - | ====Electrophoreis====
| |
| - | Sample: 1, 2, and control, Maker: 100 and lambda
| |
| - | M 1 2 C M
| |
| - | [[Image:KyotoExp100824-1.png]]
| |
| - | We found the band of sample 1 and 2 about 3000bp and there wasn't the band of sample control. So, we confirmed the PCR and RE were completed successfully.
| |
| - | ====Ligation====
| |
| - | ====Point mutation of SRRz====
| |
| - | {| class="experiments"
| |
| - | !Sample||10x||dNTPs||MgSO4||Primer1||Primer2||Template||Water||KOD-plus-||total
| |
| - | |-
| |
| - | |1||5||5||3||1.5||1.5||1||32||1||50
| |
| - | |-
| |
| - | |2||5||5||3||1.5||1.5||1||32||1||50
| |
| - | |-
| |
| - | |control||5||5||3||1.5||1.5||1||32||1||50
| |
| - | |-
| |
| - | |}
| |
| - | {|class="experiments"
| |
| - | |94℃||2min||
| |
| - | |-
| |
| - | |98℃||10sec||rowspan="3"|30cycles
| |
| - | |-
| |
| - | |55℃||30sec
| |
| - | |-
| |
| - | |68℃||4min
| |
| - | |-
| |
| - | |4℃||hold||
| |
| - | |}
| |
| - | ====Restriction Digestion(DpnI), electrophoresis and ligation====
| |
| - | [[Image:KyotoExp100824-2.png]]
| |
| - | We could find point mutation PCR and restriction enzyme of DpnI was done.
| |
| - |
| |
| - |
| |
| - | ===PCR of E0240===
| |
| - | {| class="experiments"
| |
| - | !Sample||10}||dNTPs||MgSO4||VF2||VR||Template||Water||KOD-plus-||Total
| |
| - | |-
| |
| - | |1||5||5||3||1.5||1.5||1||31.5||1||50
| |
| - | |-
| |
| - | |2||5||5||3||1.5||1.5||1||31.5||1||50
| |
| - | |-
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | ===PCR Purification===
| |
| - | Sample1: 5.5*50(ng/µL)
| |
| - | Sample2: 5.2*50(ng/µL)
| |
| - | ====Restriction Digestion(EcoRI, PstI) and Gel extraction====
| |
| - | Sample1: 28.8 (ng/µL)
| |
| - | Sample2: 26.4 (ng/µL)
| |
| - | ====Transformation====
| |
| - | Sample: rrΔTMD1GFP1. 2. control, and rSRRz1. 2. control
| |
| - |
| |
| - |
| |
| - | ===Wednesday, August 25 <span class="by">By:Ken, Tomo, Kazuya, Tasuku, Takuya<span>===
| |
| - | ====Making culture and Master plate====
| |
| - | {| class="experiments"
| |
| - | |rrΔTMD1-1||rowspan="2"|Many Colonies
| |
| - | |-
| |
| - | |rrΔTMD1-2
| |
| - | |-
| |
| - | |rrΔTMD1-C-||zero
| |
| - | |-
| |
| - | |rSRRz-1||rowspan="2"|Many Colonies
| |
| - | |-
| |
| - | |rSRRz-2
| |
| - | |-
| |
| - | |rSRRz-C-||zero
| |
| - | |}
| |
| - | ====Miniprep of 1-5G====
| |
| - | 29.0 (ng/µL)
| |
| - | ====Restriction Digestion and purification of 1-5G(low copy plasmid) and lac low====
| |
| - | {| class="experiments"
| |
| - | !Sample name||Template||10xbuffer||100xbuffer||EcoRI||SpeI||PstI||Water||Total
| |
| - | |-
| |
| - | |1-5G||50||6||0.6||0.4||0.4||-||2.6||60
| |
| - | |-
| |
| - | |Lac low||10||4||0.4||-||0.3||0.3||25||40
| |
| - | |}
| |
| - | {|class="experiments"
| |
| - | |Sample Name||Concentration(ng/µL)
| |
| - | |-
| |
| - | |1-5G||18.4
| |
| - | |-
| |
| - | |Lac low||8.6
| |
| - | |}
| |
| - | ====Ligation of <partinfo>E0240</partinfo> and <partinfo>pSB4K5</partinfo>, Transformation====
| |
| - |
| |
| - |
| |
| - | ===Thursday, August 26 <span class="by">By:Ken, Tomo, Kazuya, Tasuku, Takuya, Fumitaka</span>
| |
| - | ====Miniprep====
| |
| - | {| class="experiments"
| |
| - | |Sample name||Concentration(ng/µL)
| |
| - | |-
| |
| - | |constP(0.7)||44.5
| |
| - | |}
| |
| - | ====Restriction Digestion of constP(0.7)====
| |
| - | {| class="experiments"
| |
| - | !Template||10xbuffer||100xbuffer||SpeI||PstI||Water||Total
| |
| - | |-
| |
| - | |25||4||0.4||0.3||0.3||10||40
| |
| - | |}
| |
| - | ====Purification of constP (0.7)====
| |
| - | 49.8 ng/µL
| |
| - |
| |
| - |
| |
| - | ===Friday, August 27 <span class="by">By:Ken, Tomo, Kazuya, Fumitaka</span>===
| |
| - | ====Making master plate of E0240 low====
| |
| - | {| class="experiments"
| |
| - | |Sample Name||Concentration(ng/µL)
| |
| - | |-
| |
| - | |rrΔTMD1 1-2||20.9
| |
| - | |-
| |
| - | |rSRRz 1-1||16.4
| |
| - | |}
| |
| - | ====Restriction Digestion of rrΔTMD1 and rSRRz====
| |
| - | {| class="experiments"
| |
| - | !Sample name||Template||10xbuffer||100xbuffer||XbaI||PstI||Water||Total
| |
| - | |-
| |
| - | |rrΔTMD1 1-2||45||6||0.6||0.3||0.3||7.8||60
| |
| - | |-
| |
| - | |rSRRz 1-1||45||6||0.6||0.3||0.3||7.8||60
| |
| - | |}
| |
| - | (13:20-14:20)
| |
| - | ====Purification====
| |
| - | {|Sample Name||Concentration(ng/µL)
| |
| - | |-
| |
| - | |rrΔTMD1 1-2||44.7
| |
| - | |-
| |
| - | |rSRRz 1-1||56.1
| |
| - | |}
| |
| - | ====Lagation and transformation====
| |
| - | lacP + rrΔTMD1 1-2
| |
| - | constP (0.7) + rrΔTMD1 1-2
| |
| - | lac low + rSRRz 1-1
| |
| - |
| |
| - |
| |
| - | ===Monday, August 30 <span class="by">By: Tomonori, Kazuya, Tasuku, Ken</span>===
| |
| - | ====Making culture and Master plate====
| |
| - | {|class="experiments"
| |
| - | |lacP rrΔTMD1GFP||Many colonies
| |
| - | |-
| |
| - | |lacP rrΔTMD1GFP(control)||Some colonies
| |
| - | |-
| |
| - | |constP rrΔTMD1GFP||Many colonies
| |
| - | |-
| |
| - | |constP rrΔTMD1GFP(control)||Many colonies
| |
| - | |-
| |
| - | |lacP rSRRz low||No colony
| |
| - | |-
| |
| - | |lacP rSRRz low(control)||No colony
| |
| - | |}
| |
| - | Discussion: There ware some colonies, which emitted green light, on the plate 1. So, we cultured those colonies on master plate.
| |
| - | On the plate 5 and 6, even though we used KRX, which is able to repress lac promoter, colonies might be dead. However, we still have to do some experience so that we confirm lac promoter cannot repress enough and E. coli cannot survive.
| |
| - |
| |
| - |
| |
| - | ===Tuesday, August 31 <span class="by">By: Tomonori, Takuya Y., Kazuya, Tasuku, Takuya, Ken<span>
| |
| - | ====Miniprep====
| |
| - | {|class="experiments"
| |
| - | |constP (0.3)||48.5 (ng/µL)
| |
| - | |-
| |
| - | |lac rrΔTMD1||107.3
| |
| - | |}
| |
| - | ====RE of constP (0.3) and lac rrΔTMD1====
| |
| - | ====Gel Extraction of lac rrΔTMD1====
| |
| - | [[image:KyotoExp100831-1.png]]
| |
| - |
| |
| - | 45min
| |
| - | Discussion: There were two band at the bottom of the gel. It was too long -45min-, and insert and vector might be contaminated. But we went on next operation.
| |
| - | ====Purification of constP (0.3) and lac rrΔTMD1====
| |
| - | {|class="experiments"
| |
| - | |constP (0.3)||5.8 (ng/µL)
| |
| - | |-
| |
| - | |lac rrΔTMD1||7.8 (ng/µL)
| |
| - | |}
| |
| - | ====Ligation and transformation====
| |
| - | {|class="experiments"
| |
| - | |Insert||Vector
| |
| - | |-
| |
| - | |lac rrΔTMD1||constP (0.3)
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | ===Wednesday, September 1 <span class="by">By: Tomonori, Kazuya, Tasuku, Fumitaka, Ken</span>
| |
| - | ====Making culture and Master plate====
| |
| - | {| class="experiments"
| |
| - | |lac rrΔTMD1 constP||many colonies
| |
| - | |-
| |
| - | |lac rrΔTMD1 const (control)||many colonies
| |
| - | |}
| |
| - | Screenig PCR of lacP-rrΔTMD1GFP-constP
| |
| - | Sample: 1-13 Control: Positive (1-23L) Maker: lambda, 100
| |
| - | M 1 2 3 4 5 6 7 8 9 10 11 12 13 P M
| |
| - | [[image:KyotoExp100901.png]]
| |
| - |
| |
| - | Discussion: All of the sample except sample 10 might be self-ligation products of constP.
| |
| - | ====Miniprep====
| |
| - | {|class="experiments"
| |
| - | |rSRRz 1-1||33.8 (ng/µL)
| |
| - | |-
| |
| - | |low||56.0 (ng/µL)
| |
| - | |}
| |
| - | ====Restriction Digestion of rSRRz and low====
| |
| - | {|class="experiments"
| |
| - | !Sample name||Template||10xbuffer||100xbuffer||EcoRI||PstI||Water||Total
| |
| - | |-
| |
| - | |rSRRz||20||4||0.4||0.3||0.3||15||40
| |
| - | |-
| |
| - | |low||20||4||0.4||0.3||0.3||15||40
| |
| - | |}
| |
| - | (13:25-14:30)
| |
| - | ====Purification====
| |
| - | {|class="experiments"
| |
| - | |rSRRz||6.5 (ng/µL)
| |
| - | |-
| |
| - | |low||16.8
| |
| - | |}
| |
| - | ====Ligation and transformation====
| |
| - | Insert: rSRRz 1-1 Vector: low copy plasmid
| |
| - |
| |
| - |
| |
| - | ===Thursday, September 2 <span class="by">By: Tomonori, Tomo, Takuya, Ken<span>===
| |
| - | ====Making culture and Master plate====
| |
| - | {|class="experiments"
| |
| - | |rSRRz low||13 colonies
| |
| - | |-
| |
| - | |rSRRz low (Control)||13colonies
| |
| - | |}
| |
| - | ====Screening PCR of rSRRz low====
| |
| - | Sample: rSRRz (1-13)
| |
| - | Maker: lambda, 100
| |
| - | Control: Positive (1-23L), Neganive
| |
| - | M 1 2 3 4 5 6 7 8 9 10 11 12 13 P N M
| |
| - | [[image:KyotoExp100902.png]]
| |
| - |
| |
| - | Discussion: From sample 1, two vectors might be ligated. Sample 3 and 4, rSRRz might be inserted in low copy plasmid correctly. Sample 11, it might be the self-ligation product of low copy plasmid. Anyway, we decided to culture those 4 colonies on master plate.
| |
| - |
| |
| - |
| |
| - | ===Friday, September 3 <span class="by">By: Tomonori, Tomo, Kazuya, Tasuku, Fumitaka, Ken</span>===
| |
| - | ====Making culture====
| |
| - | lac rrΔTMD1 1, 3
| |
| - | rrΔTMD1 1-1, 1-2
| |
| - | rSRRz 1-1, 1-2
| |
| - | ML
| |
| - | ----
| |
"
