Team:Kyoto/Notebook/Construction

From 2010.igem.org

(Difference between revisions)
(New page: {{:Team:Kyoto/Header}} ==Notebook: Construction for Lysisbox== ===Tuesday, July 20 <span class="by">By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto</span>=== ====[[Team:Kyoto/Protocols#Transfo...)
Line 1: Line 1:
{{:Team:Kyoto/Header}}
{{:Team:Kyoto/Header}}
==Notebook: Construction for Lysisbox==
==Notebook: Construction for Lysisbox==
-
===Tuesday, July 20 <span class="by">By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto</span>===
+
==Tuesday, July 20 <span class="by">By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto</span>===
-
====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
+
===[[Team:Kyoto/Protocols#Transformation|Transformation]]====
{| class="experiments"
{| class="experiments"
!Name||Well||Sample||Competent Cells||Total||Plate||Incubation||Result
!Name||Well||Sample||Competent Cells||Total||Plate||Incubation||Result
Line 25: Line 25:
-
===Wednesday, July 21 <span class="by">By: Wataru, Ken, Makoto, Takuya Y.</span>===
+
==Wednesday, July 21 <span class="by">By: Wataru, Ken, Makoto, Takuya Y.</span>===
-
====Culture at 37&#x2103; from 07/21 20:50 to 07/22 17:00 and Making Master Plate====
+
===Culture at 37&#x2103; from 07/21 20:50 to 07/22 17:00 and Making Master Plate====
-
====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
+
===[[Team:Kyoto/Protocols#Transformation|Transformation]]====
{| class="experiments"
{| class="experiments"
!Name||Well||Sample||Competent Cells||Total||Plate||Incubation||Result
!Name||Well||Sample||Competent Cells||Total||Plate||Incubation||Result
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|<partinfo>B0015</partinfo>||1-23-L||1||20||21||&#x25CB;
|<partinfo>B0015</partinfo>||1-23-L||1||20||21||&#x25CB;
|}
|}
-
====[[Team:Kyoto/Protocols#Stantard PCR|PCR]] for SRRz and S====
+
===[[Team:Kyoto/Protocols#Stantard PCR|PCR]] for SRRz and S====
{| class="experiments"
{| class="experiments"
!No.||Water||MgSO4||dNTPs||10xBuffer||Template DNA||Primer Fwd.||Primer Rev. (SRRz)||Primer Rev. (S)||KOD Plus ver.2||Total
!No.||Water||MgSO4||dNTPs||10xBuffer||Template DNA||Primer Fwd.||Primer Rev. (SRRz)||Primer Rev. (S)||KOD Plus ver.2||Total
Line 69: Line 69:
-
===Thursday, July 22 <span class="by">By: Wataru</span>===
+
==Thursday, July 22 <span class="by">By: Wataru</span>===
-
====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] (40min) of the PCR Products====
+
===[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] (40min) of the PCR Products====
[[Image:KyotoExp100722-1.png|300px|right]]
[[Image:KyotoExp100722-1.png|300px|right]]
{| class="electrophoresis"
{| class="electrophoresis"
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|}
|}
Marker: 100bp, 1kb, 1kb, 100bp.
Marker: 100bp, 1kb, 1kb, 100bp.
-
====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
+
===[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
{| class="experiments"
{| class="experiments"
!Name||Concentration
!Name||Concentration
Line 109: Line 109:
|}
|}
The concentration of all samples was very week. Probably our shaking incubation was week.
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>====
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===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>===
+
==Friday, July 23 <span class="by">By: Wataru, Tomo, Makoto</span>===
-
====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
+
===[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
{| class="experiments"
{| class="experiments"
!Name||Concentration
!Name||Concentration
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|}
|}
We lost <partinfo>B0015</partinfo> by our mistake. The concentration of <partinfo>pSB4K5</partinfo> is high, so this condition of shaking incubation is moderate.
We lost <partinfo>B0015</partinfo> by our mistake. The concentration of <partinfo>pSB4K5</partinfo> is high, so this condition of shaking incubation is moderate.
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====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
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===[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
{| class="experiments"
{| class="experiments"
!No.||Name||Concentration||New Name
!No.||Name||Concentration||New Name
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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.
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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====[[Team:Kyoto/Protocols#Standard_PCR|Standard PCR]] for SRRz====
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===[[Team:Kyoto/Protocols#Standard_PCR|Standard PCR]] for SRRz====
{| class="experiments"
{| class="experiments"
!No.||Water||MgSO4||dNTPs||10xBuffer||Template DNA||Primer Fwd. (SRRz)||Primer Rev. (SRRz)||KOD plus ver.2||Total
!No.||Water||MgSO4||dNTPs||10xBuffer||Template DNA||Primer Fwd. (SRRz)||Primer Rev. (SRRz)||KOD plus ver.2||Total
Line 163: Line 163:
|4&#x2103;||forever||
|4&#x2103;||forever||
|}
|}
-
====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]] and [[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] (35min) to check function of our Restriction Enzyme====
+
===[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]] and [[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] (35min) to check function of our Restriction Enzyme====
{| class="experiments"
{| class="experiments"
!No.||Name||Sample||10xBuffer||BSA||colspan="2"|Enzyme||MilliQ||Total||Incubation
!No.||Name||Sample||10xBuffer||BSA||colspan="2"|Enzyme||MilliQ||Total||Incubation
Line 180: Line 180:
Marker: 1kb.
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.
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>====
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===[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] and [[Team:Kyoto/Protocols#Ligation|Ligation]] to insert S gene to <partinfo>E0840</partinfo>====
{| class="experiments"
{| class="experiments"
!Name||Sample||10xBuffer||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total||Incubation
!Name||Sample||10xBuffer||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total||Incubation
Line 200: Line 200:
-
===Monday, July 26 <span class="by">By: Wataru, Tomonori, Makoto</span>===
+
==Monday, July 26 <span class="by">By: Wataru, Tomonori, Makoto</span>===
-
====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of PCR Products====
+
===[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of PCR Products====
[[Image:KyotoExp100726-1.png|300px|right]]
[[Image:KyotoExp100726-1.png|300px|right]]
{| class="electrophoresis"
{| class="electrophoresis"
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Marker: 1kb.
Marker: 1kb.
At the condition 4 (4.5&micro;l MgSO4) and 6 (6&micro;l MgSO4), SRRz is amplified very much. So we decided to use them.
At the condition 4 (4.5&micro;l MgSO4) and 6 (6&micro;l MgSO4), SRRz is amplified very much. So we decided to use them.
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====PCR Purification====
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===PCR Purification====
{| class="experiments"
{| class="experiments"
!No.||Name||Concentration||New Name
!No.||Name||Concentration||New Name
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|6||SRRZ||59.6||SRRz<sub>Sam7</sub>(2)
|6||SRRZ||59.6||SRRz<sub>Sam7</sub>(2)
|}
|}
-
====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
+
===[[Team:Kyoto/Protocols#Transformation|Transformation]]====
{| class="experiments"
{| class="experiments"
!Name||Well||Sample||Competent Cell||Total||Plate||Incubation||Result
!Name||Well||Sample||Competent Cell||Total||Plate||Incubation||Result
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|<partinfo>J04450</partinfo>||1-5-E||1||20||21||&#xD7;
|<partinfo>J04450</partinfo>||1-5-E||1||20||21||&#xD7;
|}
|}
-
====Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>====
+
===Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>====
-
===Tuesday, July 27 <span class="by">By: Wataru, Tomo, Kazuya, Ken, Naoi===
+
==Tuesday, July 27 <span class="by">By: Wataru, Tomo, Kazuya, Ken, Naoi===
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====[[Team:Kyoto/Protocols#Colony PCR|Colony PCR]] of S<sub>Sam7</sub>-E0840 (Electrophoresis for 35min)====
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===[[Team:Kyoto/Protocols#Colony PCR|Colony PCR]] of S<sub>Sam7</sub>-E0840 (Electrophoresis for 35min)====
[[Image:KyotoExp100727-1.png|300px|right]]
[[Image:KyotoExp100727-1.png|300px|right]]
{| class="electrophoresis"
{| class="electrophoresis"
Line 280: Line 280:
|}
|}
Marker: 1kb, 100bp
Marker: 1kb, 100bp
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====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
+
===[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
{| class="experiments"
{| class="experiments"
!Name||Concentration
!Name||Concentration
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|<partinfo>E0840</partinfo>||120.1
|<partinfo>E0840</partinfo>||120.1
|}
|}
-
====[[Team:Kyoto/Protocols#RestrictionDigestion|Restriction Digestion]]====
+
===[[Team:Kyoto/Protocols#RestrictionDigestion|Restriction Digestion]]====
{|class="experiments"
{|class="experiments"
!||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total||Incubation
!||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total||Incubation
Line 300: Line 300:
|SRRz<sub>Sam7</sub>(2)||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#Ligation|Ligation]]====
-
====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
+
===[[Team:Kyoto/Protocols#Transformation|Transformation]]====
{| class="experiments"
{| class="experiments"
!Name||Sample||Competent Cells||Total||Plate||Incubation||Result
!Name||Sample||Competent Cells||Total||Plate||Incubation||Result
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-
===Wednesday, July 28 <span class="by">By: </span>===
+
==Wednesday, July 28 <span class="by">By: </span>===
-
====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
+
===[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
{| class="experiments"
{| class="experiments"
!Name||Concentration
!Name||Concentration
Line 321: Line 321:
|}
|}
Diluted S<sub>Sam7</sub>(1)-E0840 and S<sub>Sam7</sub>(2)-E0840 20 times with water, and used as template DNA.
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====
+
===[[Team:Kyoto/Protocols#Mutagenesis (Point mutation, Deletion, Insertion)|Deletion PCR]] to delete a functional domain of S gene====
{| class="experiments"
{| 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
!||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
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|4&#x2103;||forever||
|4&#x2103;||forever||
|}
|}
-
====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] to check the function of DpnI====
+
===[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]] to check the function of DpnI====
{| class="experiments"
{| class="experiments"
!Name||Sample||fast digestion buffer||DpnI||MilliQ||Total
!Name||Sample||fast digestion buffer||DpnI||MilliQ||Total
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|S<sub>Sam7,&Delta;TMD1</sub>(2)-E0840 (2)||3||1||0.1||5.8||10
|S<sub>Sam7,&Delta;TMD1</sub>(2)-E0840 (2)||3||1||0.1||5.8||10
|}
|}
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====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] for 35min====
+
===[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] for 35min====
[[image:KyotoExp100728-1.png|300px|right]]
[[image:KyotoExp100728-1.png|300px|right]]
{| class="electrophoresis"
{| class="electrophoresis"
Line 369: Line 369:
-
===Thursday, July 29 <span class="by">By: </span>===
+
==Thursday, July 29 <span class="by">By: </span>===
-
====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]]====
+
===[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]]====
{| class="experiments"
{| class="experiments"
!Name||Sample volume||Fastdigestion Buffer||colspan="2"|Enzyme 1||MilliQ||Total||Incubation
!Name||Sample volume||Fastdigestion Buffer||colspan="2"|Enzyme 1||MilliQ||Total||Incubation
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|S<sub>Sam7,&Delta;TMD1</sub>(2)-E0840 (1)||50||6||DpnI||0.2||3.8||60
|S<sub>Sam7,&Delta;TMD1</sub>(2)-E0840 (1)||50||6||DpnI||0.2||3.8||60
|}
|}
-
====[[Team:Kyoto/Protocols#Ligation|Ligation]] and [[Team:Kyoto/Protocols#Phosphorylation|Phosphorylation]]====
+
===[[Team:Kyoto/Protocols#Ligation|Ligation]] and [[Team:Kyoto/Protocols#Phosphorylation|Phosphorylation]]====
{| class="experiments"
{| class="experiments"
!Name||Sample||MilliQ||Ligation High||T4 Kinase||Total||Incubation
!Name||Sample||MilliQ||Ligation High||T4 Kinase||Total||Incubation
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|S<sub>Sam7,&Delta;TMD1</sub>(1)-E0840 (2)||2||7||5||1||15
|S<sub>Sam7,&Delta;TMD1</sub>(1)-E0840 (2)||2||7||5||1||15
|}
|}
-
====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
+
===[[Team:Kyoto/Protocols#Transformation|Transformation]]====
{| class="experiments"
{| class="experiments"
!Name||Sample Volume||Competent Cell||Total||Plate||Incubation||Result
!Name||Sample Volume||Competent Cell||Total||Plate||Incubation||Result
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-
===Monday, August 2 <span class="by">By: Wataru, Ken</span>===
+
==Monday, August 2 <span class="by">By: Wataru, Ken</span>===
-
====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
+
===[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
{|class="experiments"
{|class="experiments"
!Name||Concentration(ng/&micro;L)
!Name||Concentration(ng/&micro;L)
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|<partinfo>R0011</partinfo>||18.6
|<partinfo>R0011</partinfo>||18.6
|}
|}
-
====[[Team:Kyoto/Protocols#Standard_PCR|Standard PCR]] of <partinfo>E0240</partinfo>====
+
===[[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.
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"
{|class="experiments"
Line 431: Line 431:
|4&#x2103;||forever||
|4&#x2103;||forever||
|}
|}
-
====[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]]====
+
===[[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]]====
-
====PCR Purification====
+
===PCR Purification====
{|class="experiments"
{|class="experiments"
!Sample number||Concentration(ng/&micro;L)
!Sample number||Concentration(ng/&micro;L)
Line 440: Line 440:
|E0240<sub>2</sub>||55.3
|E0240<sub>2</sub>||55.3
|}
|}
-
====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]] for inserting <partinfo>E0240</partinfo> to pSB4K5 by 3A assembly====
+
===[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]] for inserting <partinfo>E0240</partinfo> to pSB4K5 by 3A assembly====
{| class="experiments"
{| class="experiments"
!Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
!Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
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|E0240<sub>2</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====
+
===PCR Purification====
{| class="experiments"
{| class="experiments"
!Name||Concentration(ng/&micro;L)||Volume(&micro;L)
!Name||Concentration(ng/&micro;L)||Volume(&micro;L)
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|}
|}
Stored at -20&#x2103;.
Stored at -20&#x2103;.
-
====Error PCR====
+
===Error PCR====
{|class="experiments"
{|class="experiments"
!Name||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10&micro;M)||Primer VR(10&micro;M)||Template &Delta;1||Template||Template||KOD Pllus ver.2||Total
!Name||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10&micro;M)||Primer VR(10&micro;M)||Template &Delta;1||Template||Template||KOD Pllus ver.2||Total
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|4&#x2103;||forever||
|4&#x2103;||forever||
|}
|}
-
====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
+
===[[Team:Kyoto/Protocols#Transformation|Transformation]]====
{| class="experiments"
{| class="experiments"
!Name||Sample (&micro;l)||Competent Cells (&micro;l)||Total (&micro;l)||Plate||Incubation||Result
!Name||Sample (&micro;l)||Competent Cells (&micro;l)||Total (&micro;l)||Plate||Incubation||Result
Line 488: Line 488:
-
===Tuesday, August 3 <span class="by">By: </span>===
+
==Tuesday, August 3 <span class="by">By: </span>===
-
====Culture of each two colonies of S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-1 and S<sub>&Delta;TMD1</sub>-E0840<sub>2</sub> for 37&#x2103; 08/03-08/04====
+
===Culture of each two colonies of S<sub>&Delta;TMD1</sub>-E0840<sub>1</sub>-1 and S<sub>&Delta;TMD1</sub>-E0840<sub>2</sub> for 37&#x2103; 08/03-08/04====
-
====[[Team:Kyoto/Protocols#Miniprep|Miniprep]] for Construction of Measure(''lac''P) and Measure(Standard)====
+
===[[Team:Kyoto/Protocols#Miniprep|Miniprep]] for Construction of Measure(''lac''P) and Measure(Standard)====
{|class="experiments"
{|class="experiments"
!Sample number||Concentration(ng/&micro;L)
!Sample number||Concentration(ng/&micro;L)
Line 498: Line 498:
|<partinfo>R0011</partinfo>||26.8
|<partinfo>R0011</partinfo>||26.8
|}
|}
-
====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]]====
+
===[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]]====
{|class="experiments"
{|class="experiments"
!Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
!Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
Line 510: Line 510:
|E0240<sub>2</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====
+
===PCR Purification====
{|class="experiments"
{|class="experiments"
!Sample number||Concentration(ng/&micro;L)
!Sample number||Concentration(ng/&micro;L)
Line 521: Line 521:
|}
|}
pSB4K5(E-P) is concentrated 10&micro;L and E0240<sub>1</sub>(X-P), E0240<sub>2</sub>(X-P) are concentrated 1&micro;L.
pSB4K5(E-P) is concentrated 10&micro;L and E0240<sub>1</sub>(X-P), E0240<sub>2</sub>(X-P) are concentrated 1&micro;L.
-
====Ethanol Precipitation====
+
===Ethanol Precipitation====
-
====Dilution of <partinfo>pSB4K5</partinfo> by 2&micro;l MilliQ====
+
===Dilution of <partinfo>pSB4K5</partinfo> by 2&micro;l MilliQ====
-
====[[Team:Kyoto/Protocols#Ligation|Ligation]]====
+
===[[Team:Kyoto/Protocols#Ligation|Ligation]]====
{|class="experiments"
{|class="experiments"
!||Vector||colspan="2"|Insert 1||colspan="2"|Insert 2||Ligation High||Total||Incubation
!||Vector||colspan="2"|Insert 1||colspan="2"|Insert 2||Ligation High||Total||Incubation
Line 531: Line 531:
|R0011-E0240<sub>2</sub>[Low]||pSB4K5(E-P)||1||R0011(E-S)||1||E0240<sub>2</sub>(X-P)||1||3||15
|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====
+
===[[Team:Kyoto/Protocols#Standard PCR|Standard PCR]] of <partinfo>J23101</partinfo>-<partinfo>E0240</partinfo> that is important in the measurement of RPU====
{|class="experiments"
{|class="experiments"
!Name||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10&micro;M)||Primer VR(10&micro;M)||Template J23101-E0240||KOD plus ver.2 ||Total
!Name||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10&micro;M)||Primer VR(10&micro;M)||Template J23101-E0240||KOD plus ver.2 ||Total
Line 550: Line 550:
|4&#x2103;||forever||
|4&#x2103;||forever||
|}
|}
-
====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
+
===[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
{|class="experiments"
{|class="experiments"
!Name||Concentration(ng/&micro;L)
!Name||Concentration(ng/&micro;L)
Line 556: Line 556:
|J23101-E0240||40.6
|J23101-E0240||40.6
|}
|}
-
====[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]====
+
===[[Team:Kyoto/Protocols#Restriction Digestion|Restriction Digestion]]====
{|class="experiments"
{|class="experiments"
!Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
!Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
Line 562: Line 562:
|J23101-E0240(E-P)||45||6||0.6||EcoRI||0.2||PstI||0.2||8||60
|J23101-E0240(E-P)||45||6||0.6||EcoRI||0.2||PstI||0.2||8||60
|}
|}
-
====[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
+
===[[Team:Kyoto/Protocols#PCR Purification|PCR Purification]]====
{| class="experiments"
{| class="experiments"
!Name||Concentration(ng/&micro;L)||Volume(&micro;L)
!Name||Concentration(ng/&micro;L)||Volume(&micro;L)
Line 569: Line 569:
|}
|}
J23101-E0240(E-P) is concentrated 7&micro;L
J23101-E0240(E-P) is concentrated 7&micro;L
-
====[[Team:Kyoto/Protocols#Ligation|Ligation]]====
+
===[[Team:Kyoto/Protocols#Ligation|Ligation]]====
{|class="experiments"
{|class="experiments"
!||Vector||colspan="2"|Insert||colspan="2"|Ligation High||Total||Incubation
!||Vector||colspan="2"|Insert||colspan="2"|Ligation High||Total||Incubation
Line 576: Line 576:
|}
|}
<div class="measure-construction"><br />
<div class="measure-construction"><br />
-
====Transformation====
+
===Transformation====
{| class="experiments"
{| class="experiments"
!Name||Conc(/&micro;L)||Sample Volume(&micro;L)||Competent Cell(&micro;L)||Total||Plate||Incubation
!Name||Conc(/&micro;L)||Sample Volume(&micro;L)||Competent Cell(&micro;L)||Total||Plate||Incubation
Line 588: Line 588:
-
===Thursday, August 5 <span class="by">By: </span>===
+
==Thursday, August 5 <span class="by">By: </span>===
-
====Result of Transformation====
+
===Result of Transformation====
{|class="experiments"
{|class="experiments"
|R0011-E0240<sub>1</sub>[Low]||rowspan="3"|Many colonies
|R0011-E0240<sub>1</sub>[Low]||rowspan="3"|Many colonies
Line 639: Line 639:
-
===Friday, August 6===
+
==Friday, August 6===
-
====Miniprep====
+
===Miniprep====
{| class="experiments"
{| class="experiments"
!Name
!Name
Line 666: Line 666:
|J23101-E0240[Low]-3
|J23101-E0240[Low]-3
|}
|}
-
====Restriction Digestion====
+
===Restriction Digestion====
{|class="experiments"
{|class="experiments"
!Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
!Name||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
Line 730: Line 730:
[[Image:KyotoExp100806-1.png]]
[[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.
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)====
+
===Error PCR (Retry)====
{| class="experiments"
{| class="experiments"
!Name||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10&micro;M)||Primer VR(10&micro;M)||Template &Delta;TMD failed(50ng/&micro;L)||KOD plus ver.2||Total
!Name||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10&micro;M)||Primer VR(10&micro;M)||Template &Delta;TMD failed(50ng/&micro;L)||KOD plus ver.2||Total
Line 751: Line 751:
|4&#x2103;||forever||
|4&#x2103;||forever||
|}
|}
-
====Transformation====
+
===Transformation====
{| class="experiments"
{| class="experiments"
!Name||Conc(/&micro;L)||Sample Volum(&micro;L)||Competent Cell(&micro;L)||Total||Plate||Incubation
!Name||Conc(/&micro;L)||Sample Volum(&micro;L)||Competent Cell(&micro;L)||Total||Plate||Incubation
Line 765: Line 765:
-
===Monday, August 9 <span class="by">By: Wataru, Tomonori, Ken, Takuya</span>===
+
==Monday, August 9 <span class="by">By: Wataru, Tomonori, Ken, Takuya</span>===
-
====Miniprep of MS and ML====
+
===Miniprep of MS and ML====
{|class="experiments"
{|class="experiments"
!Sample number||concentration(ng/&micro;L)
!Sample number||concentration(ng/&micro;L)
Line 774: Line 774:
|ML||146.6
|ML||146.6
|}
|}
-
====Transfotrmation of MS and ML====
+
===Transfotrmation of MS and ML====
{|class="experiments"
{|class="experiments"
!Sample||conc(ng/&micro;L)||Sample vol(&micro;L)||Competent Cell||Competent cell vol(&micro;L)||Total vol(&micro;L)||Plate||Incuvation
!Sample||conc(ng/&micro;L)||Sample vol(&micro;L)||Competent Cell||Competent cell vol(&micro;L)||Total vol(&micro;L)||Plate||Incuvation
Line 782: Line 782:
|ML||146.6||2||KRX||50||52
|ML||146.6||2||KRX||50||52
|}
|}
-
====Restriction enzyme digestion and ethanol precipitation====
+
===Restriction enzyme digestion and ethanol precipitation====
To use lac p for next ligation, we digested 1-6-G by EroRI and PstI
To use lac p for next ligation, we digested 1-6-G by EroRI and PstI
{|class="experiments"
{|class="experiments"
Line 791: Line 791:
Incubate 37&#x2103; 8/9 16:20‾18:20
Incubate 37&#x2103; 8/9 16:20‾18:20
After restriction enzyme digestion, we did ethanol precipitation.
After restriction enzyme digestion, we did ethanol precipitation.
-
====Ligation and Transformation====
+
===Ligation and Transformation====
{|class="experiments"
{|class="experiments"
!Sample||Conc (nu/&micro;L)||Sample vol (&micro;L)||Competent cell||Competent cell vol (&micro;L)||Total vol (&micro;L)||Plate||Incuvation
!Sample||Conc (nu/&micro;L)||Sample vol (&micro;L)||Competent cell||Competent cell vol (&micro;L)||Total vol (&micro;L)||Plate||Incuvation
Line 801: Line 801:
-
===Tuesday, August 10 <span class="by">By: Wataru, Tomonori, Ken, Fumitaka</span>
+
==Tuesday, August 10 <span class="by">By: Wataru, Tomonori, Ken, Fumitaka</span>
-
====Making culture plate on lac p (low), MS and ML====
+
===Making culture plate on lac p (low), MS and ML====
{|class="experiments"
{|class="experiments"
|rowspan="2"|Lac p (low)||KRX||rowspan="4"|Many colonies
|rowspan="2"|Lac p (low)||KRX||rowspan="4"|Many colonies
Line 812: Line 812:
|ML||KRX
|ML||KRX
|}
|}
-
====Minprep of &Delta;TMD1+GFP====
+
===Minprep of &Delta;TMD1+GFP====
{|class="experiments"
{|class="experiments"
!Sample number||Concentration (ng/&micro;L)
!Sample number||Concentration (ng/&micro;L)
Line 825: Line 825:
|}
|}
37&#x2103; 8/9 18:00‾8/10 9:00
37&#x2103; 8/9 18:00‾8/10 9:00
-
====Culture and Master Plate====
+
===Culture and Master Plate====
-
===Wednesday, August 11 <span class="by">By: Wataru, Naoi, Ken, Takuya</span>
+
==Wednesday, August 11 <span class="by">By: Wataru, Naoi, Ken, Takuya</span>
{| class="experiments"
{| class="experiments"
!Sample||Medium||Cloud||Incubation
!Sample||Medium||Cloud||Incubation
Line 861: Line 861:
|}
|}
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.
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'====
+
===Miniprep of C2+lac(low), S-R-Rz 1', 3'====
lac(low)1 : 31.2 (ng/&micro;L)
lac(low)1 : 31.2 (ng/&micro;L)
lac(low)2 : 29.9 (ng/&micro;L)
lac(low)2 : 29.9 (ng/&micro;L)
-
====Restriction Digestion and electrophoresis of lac (low) 1 and 3====
+
===Restriction Digestion and electrophoresis of lac (low) 1 and 3====
{| class="experiments"
{| class="experiments"
!Name||EcoRI||PstI
!Name||EcoRI||PstI
Line 880: Line 880:
[[image:KyotoExp100811-1.png]]
[[image:KyotoExp100811-1.png]]
Discussion: Each enzyme correctly cut samples.
Discussion: Each enzyme correctly cut samples.
-
====Screening PCR of SRRz====
+
===Screening PCR of SRRz====
Sample: 1-20
Sample: 1-20
Control: P(1-23L) P'(2-8E) N
Control: P(1-23L) P'(2-8E) N
Line 891: Line 891:
-
===Thursday, August 12 <span class="by">By: Wataru, Ken</span>===
+
==Thursday, August 12 <span class="by">By: Wataru, Ken</span>===
-
====Restriction Digestion and electrophoresis of <partinfo>B0015</partinfo>====
+
===Restriction Digestion and electrophoresis of <partinfo>B0015</partinfo>====
{| class="experiments"
{| class="experiments"
!Sample name||Template||10xbuffer||100xbuffer||EcoRI||XbaI 1||XbaI 2||SpeI||PstI 1||PstI 2||Water||Total
!Sample name||Template||10xbuffer||100xbuffer||EcoRI||XbaI 1||XbaI 2||SpeI||PstI 1||PstI 2||Water||Total
Line 916: Line 916:
-
===Thursday, August 19 <span class="by">By: Wataru, Tomo, Ken</span>
+
==Thursday, August 19 <span class="by">By: Wataru, Tomo, Ken</span>
-
====Miniprep of S&Delta;TMD1GFP====
+
===Miniprep of S&Delta;TMD1GFP====
29.6(ng/&micro;g)
29.6(ng/&micro;g)
-
====Point mutation PCR of &Delta;TMD1GFP====
+
===Point mutation PCR of &Delta;TMD1GFP====
{| class="experiments"
{| class="experiments"
!Sample number||Template||10xbuffer||dNTPs||MgSO4||Primer 1||Primer 2||Water||KOD-plus-||Total
!Sample number||Template||10xbuffer||dNTPs||MgSO4||Primer 1||Primer 2||Water||KOD-plus-||Total
Line 940: Line 940:
|4.0||forever||
|4.0||forever||
|}
|}
-
====Restriction Digestion(DpnI): 17:50-18:50====
+
===Restriction Digestion(DpnI): 17:50-18:50====
-
====Electrophoresis====
+
===Electrophoresis====
Sample: 1, 2, Control Marker: lambda, 100
Sample: 1, 2, Control Marker: lambda, 100
[[Image:KyotoExp100819-1.png]]
[[Image:KyotoExp100819-1.png]]
-
====Ligation and Transformation====
+
===Ligation and Transformation====
We named point mutation PCR products r&Delta;TMD1GFP.
We named point mutation PCR products r&Delta;TMD1GFP.
-
===Monday, August 23 <span class="by">By: Wataru, Tomo, Ken, Fumitaka, Tasuku</span>===
+
==Monday, August 23 <span class="by">By: Wataru, Tomo, Ken, Fumitaka, Tasuku</span>===
-
====Miniprep of &Delta;TMD1====
+
===Miniprep of &Delta;TMD1====
{| class="experiments"
{| class="experiments"
|Sample number||Concentration(ng/&micro;g)
|Sample number||Concentration(ng/&micro;g)
Line 957: Line 957:
|2-2||49.9
|2-2||49.9
|}
|}
-
====Sequencing of &Delta;TMD1 and MS====
+
===Sequencing of &Delta;TMD1 and MS====
Sample: r&delta;TMD1GFP1-1, 2-2, and MS
Sample: r&delta;TMD1GFP1-1, 2-2, and MS
Discussion: The sequencing was in success and the results were desirable. It meant point mutation of &delta;TMD1GFP was succeeded and sequence of MS was confirmed. We decided to use r&delta;TMD1GFP.
Discussion: The sequencing was in success and the results were desirable. It meant point mutation of &delta;TMD1GFP was succeeded and sequence of MS was confirmed. We decided to use r&delta;TMD1GFP.
-
====Screening PCR of SRRz-DT====
+
===Screening PCR of SRRz-DT====
Sample: 1-13, Marker: lambda and 100, Control:P(1-23L) and N
Sample: 1-13, Marker: lambda and 100, Control:P(1-23L) and N
{| class="experiments"
{| class="experiments"
Line 978: Line 978:
[[Image:KyotoExp100823-1.png]]
[[Image:KyotoExp100823-1.png]]
Discussion: We found the band; about 200bp, and it meant the lligation was completed successfully.
Discussion: We found the band; about 200bp, and it meant the lligation was completed successfully.
-
====Deletion PCR of r&Delta;TMD1GFP 2-2====
+
===Deletion PCR of r&Delta;TMD1GFP 2-2====
{| class="experiments"
{| class="experiments"
!Sample||10x||dNTPs||Primer1||Primer2||Template||Water||KOD-plus-||Total
!Sample||10x||dNTPs||Primer1||Primer2||Template||Water||KOD-plus-||Total
Line 999: Line 999:
|4&#x2103;||hold||
|4&#x2103;||hold||
|}
|}
-
====Restriction Digestion(DpnI)====
+
===Restriction Digestion(DpnI)====
{| class="experiments"
{| class="experiments"
|Template||25(&micro;L)
|Template||25(&micro;L)
Line 1,008: Line 1,008:
|}
|}
19:10-20:10
19:10-20:10
-
====Ligation====
+
===Ligation====
{|class="experiments"
{|class="experiments"
!Sample||Template||Water||Ligation high||T4 Kinase||total
!Sample||Template||Water||Ligation high||T4 Kinase||total
Line 1,019: Line 1,019:
|}
|}
20:15-21:15
20:15-21:15
-
====Transformation====
+
===Transformation====
We named sample 1, 2 and control rr&delta;TMD1GFP1, 2 and control.
We named sample 1, 2 and control rr&delta;TMD1GFP1, 2 and control.
-
===Tuesday, August 24 <span class="by">By:Ken, Tomo, Tasuku, Takuya</span>===
+
==Tuesday, August 24 <span class="by">By:Ken, Tomo, Tasuku, Takuya</span>===
-
====Retry of deletion PCR of r&delta;TMD1 GFP====
+
===Retry of deletion PCR of r&delta;TMD1 GFP====
{| class="experiments"
{| class="experiments"
!Sample||10x||dNTPs||MgSO4||Primer1||Primer2||Template||Water||KOD-plus-||Total
!Sample||10x||dNTPs||MgSO4||Primer1||Primer2||Template||Water||KOD-plus-||Total
Line 1,045: Line 1,045:
|4&#x2103;||hold||
|4&#x2103;||hold||
|}
|}
-
====Restriction Digestion (DpnI)====
+
===Restriction Digestion (DpnI)====
14:15-15:15
14:15-15:15
-
====Electrophoreis====
+
===Electrophoreis====
Sample: 1, 2, and control, Maker: 100 and lambda
Sample: 1, 2, and control, Maker: 100 and lambda
M    1  2  C        M
M    1  2  C        M
[[Image:KyotoExp100824-1.png]]
[[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.
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====
+
===Ligation====
-
====Point mutation of SRRz====
+
===Point mutation of SRRz====
{| class="experiments"
{| class="experiments"
!Sample||10x||dNTPs||MgSO4||Primer1||Primer2||Template||Water||KOD-plus-||total
!Sample||10x||dNTPs||MgSO4||Primer1||Primer2||Template||Water||KOD-plus-||total
Line 1,075: Line 1,075:
|4&#x2103;||hold||
|4&#x2103;||hold||
|}
|}
-
====Restriction Digestion(DpnI), electrophoresis and ligation====
+
===Restriction Digestion(DpnI), electrophoresis and ligation====
[[Image:KyotoExp100824-2.png]]
[[Image:KyotoExp100824-2.png]]
We could find point mutation PCR and restriction enzyme of DpnI was done.
We could find point mutation PCR and restriction enzyme of DpnI was done.
-
===PCR of E0240===
+
==PCR of E0240===
{| class="experiments"
{| class="experiments"
!Sample||10&#x7D;||dNTPs||MgSO4||VF2||VR||Template||Water||KOD-plus-||Total
!Sample||10&#x7D;||dNTPs||MgSO4||VF2||VR||Template||Water||KOD-plus-||Total
Line 1,091: Line 1,091:
-
===PCR Purification===
+
==PCR Purification===
Sample1: 5.5*50(ng/&micro;L)
Sample1: 5.5*50(ng/&micro;L)
Sample2: 5.2*50(ng/&micro;L)
Sample2: 5.2*50(ng/&micro;L)
-
====Restriction Digestion(EcoRI, PstI) and Gel extraction====
+
===Restriction Digestion(EcoRI, PstI) and Gel extraction====
Sample1: 28.8 (ng/&micro;L)
Sample1: 28.8 (ng/&micro;L)
Sample2: 26.4 (ng/&micro;L)
Sample2: 26.4 (ng/&micro;L)
-
====Transformation====
+
===Transformation====
Sample: rr&Delta;TMD1GFP1. 2. control, and rSRRz1. 2. control
Sample: rr&Delta;TMD1GFP1. 2. control, and rSRRz1. 2. control
-
===Wednesday, August 25 <span class="by">By:Ken, Tomo, Kazuya, Tasuku, Takuya<span>===
+
==Wednesday, August 25 <span class="by">By:Ken, Tomo, Kazuya, Tasuku, Takuya<span>===
-
====Making culture and Master plate====
+
===Making culture and Master plate====
{| class="experiments"
{| class="experiments"
|rr&Delta;TMD1-1||rowspan="2"|Many Colonies
|rr&Delta;TMD1-1||rowspan="2"|Many Colonies
Line 1,116: Line 1,116:
|rSRRz-C-||zero
|rSRRz-C-||zero
|}
|}
-
====Miniprep of 1-5G====
+
===Miniprep of 1-5G====
29.0 (ng/&micro;L)
29.0 (ng/&micro;L)
-
====Restriction Digestion and purification of 1-5G(low copy plasmid) and lac low====
+
===Restriction Digestion and purification of 1-5G(low copy plasmid) and lac low====
{| class="experiments"
{| class="experiments"
!Sample name||Template||10xbuffer||100xbuffer||EcoRI||SpeI||PstI||Water||Total
!Sample name||Template||10xbuffer||100xbuffer||EcoRI||SpeI||PstI||Water||Total
Line 1,133: Line 1,133:
|Lac low||8.6
|Lac low||8.6
|}
|}
-
====Ligation of <partinfo>E0240</partinfo> and <partinfo>pSB4K5</partinfo>, Transformation====
+
===Ligation of <partinfo>E0240</partinfo> and <partinfo>pSB4K5</partinfo>, Transformation====
-
===Thursday, August 26 <span class="by">By:Ken, Tomo, Kazuya, Tasuku, Takuya, Fumitaka</span>
+
==Thursday, August 26 <span class="by">By:Ken, Tomo, Kazuya, Tasuku, Takuya, Fumitaka</span>
-
====Miniprep====
+
===Miniprep====
{| class="experiments"
{| class="experiments"
|Sample name||Concentration(ng/&micro;L)
|Sample name||Concentration(ng/&micro;L)
Line 1,143: Line 1,143:
|constP(0.7)||44.5
|constP(0.7)||44.5
|}
|}
-
====Restriction Digestion of constP(0.7)====
+
===Restriction Digestion of constP(0.7)====
{| class="experiments"
{| class="experiments"
!Template||10xbuffer||100xbuffer||SpeI||PstI||Water||Total
!Template||10xbuffer||100xbuffer||SpeI||PstI||Water||Total
Line 1,149: Line 1,149:
|25||4||0.4||0.3||0.3||10||40
|25||4||0.4||0.3||0.3||10||40
|}
|}
-
====Purification of constP (0.7)====
+
===Purification of constP (0.7)====
49.8 ng/&micro;L
49.8 ng/&micro;L
-
===Friday, August 27 <span class="by">By:Ken, Tomo, Kazuya, Fumitaka</span>===
+
==Friday, August 27 <span class="by">By:Ken, Tomo, Kazuya, Fumitaka</span>===
-
====Making master plate of E0240 low====
+
===Making master plate of E0240 low====
{| class="experiments"
{| class="experiments"
|Sample Name||Concentration(ng/&micro;L)
|Sample Name||Concentration(ng/&micro;L)
Line 1,162: Line 1,162:
|rSRRz 1-1||16.4
|rSRRz 1-1||16.4
|}
|}
-
====Restriction Digestion of rr&Delta;TMD1 and rSRRz====
+
===Restriction Digestion of rr&Delta;TMD1 and rSRRz====
{| class="experiments"
{| class="experiments"
!Sample name||Template||10xbuffer||100xbuffer||XbaI||PstI||Water||Total
!Sample name||Template||10xbuffer||100xbuffer||XbaI||PstI||Water||Total
Line 1,171: Line 1,171:
|}
|}
(13:20-14:20)
(13:20-14:20)
-
====Purification====
+
===Purification====
{|Sample Name||Concentration(ng/&micro;L)
{|Sample Name||Concentration(ng/&micro;L)
|-
|-
Line 1,178: Line 1,178:
|rSRRz 1-1||56.1
|rSRRz 1-1||56.1
|}
|}
-
====Lagation and transformation====
+
===Lagation and transformation====
lacP + rr&Delta;TMD1 1-2
lacP + rr&Delta;TMD1 1-2
constP (0.7) + rr&Delta;TMD1 1-2
constP (0.7) + rr&Delta;TMD1 1-2
Line 1,184: Line 1,184:
-
===Monday, August 30 <span class="by">By: Tomonori, Kazuya, Tasuku, Ken</span>===
+
==Monday, August 30 <span class="by">By: Tomonori, Kazuya, Tasuku, Ken</span>===
-
====Making culture and Master plate====
+
===Making culture and Master plate====
{|class="experiments"
{|class="experiments"
|lacP rr&Delta;TMD1GFP||Many colonies
|lacP rr&Delta;TMD1GFP||Many colonies
Line 1,203: Line 1,203:
-
===Tuesday, August 31 <span class="by">By: Tomonori, Takuya Y., Kazuya, Tasuku, Takuya, Ken<span>
+
==Tuesday, August 31 <span class="by">By: Tomonori, Takuya Y., Kazuya, Tasuku, Takuya, Ken<span>
-
====Miniprep====
+
===Miniprep====
{|class="experiments"
{|class="experiments"
|constP (0.3)||48.5 (ng/&micro;L)
|constP (0.3)||48.5 (ng/&micro;L)
Line 1,210: Line 1,210:
|lac rr&Delta;TMD1||107.3
|lac rr&Delta;TMD1||107.3
|}
|}
-
====RE of constP (0.3) and lac rr&Delta;TMD1====
+
===RE of constP (0.3) and lac rr&Delta;TMD1====
-
====Gel Extraction of lac rr&Delta;TMD1====
+
===Gel Extraction of lac rr&Delta;TMD1====
[[image:KyotoExp100831-1.png]]
[[image:KyotoExp100831-1.png]]
   
   
45min
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.
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 rr&Delta;TMD1====
+
===Purification of constP (0.3) and lac rr&Delta;TMD1====
{|class="experiments"
{|class="experiments"
|constP (0.3)||5.8 (ng/&micro;L)
|constP (0.3)||5.8 (ng/&micro;L)
Line 1,222: Line 1,222:
|lac rr&Delta;TMD1||7.8 (ng/&micro;L)
|lac rr&Delta;TMD1||7.8 (ng/&micro;L)
|}
|}
-
====Ligation and transformation====
+
===Ligation and transformation====
{|class="experiments"
{|class="experiments"
|Insert||Vector
|Insert||Vector
Line 1,230: Line 1,230:
-
===Wednesday, September 1 <span class="by">By: Tomonori, Kazuya, Tasuku, Fumitaka, Ken</span>
+
==Wednesday, September 1 <span class="by">By: Tomonori, Kazuya, Tasuku, Fumitaka, Ken</span>
-
====Making culture and Master plate====
+
===Making culture and Master plate====
{| class="experiments"
{| class="experiments"
|lac rr&Delta;TMD1 constP||many colonies
|lac rr&Delta;TMD1 constP||many colonies
Line 1,243: Line 1,243:
   
   
Discussion: All of the sample except sample 10 might be self-ligation products of constP.
Discussion: All of the sample except sample 10 might be self-ligation products of constP.
-
====Miniprep====
+
===Miniprep====
{|class="experiments"
{|class="experiments"
|rSRRz 1-1||33.8 (ng/&micro;L)
|rSRRz 1-1||33.8 (ng/&micro;L)
Line 1,249: Line 1,249:
|low||56.0 (ng/&micro;L)
|low||56.0 (ng/&micro;L)
|}
|}
-
====Restriction Digestion of rSRRz and low====
+
===Restriction Digestion of rSRRz and low====
{|class="experiments"
{|class="experiments"
!Sample name||Template||10xbuffer||100xbuffer||EcoRI||PstI||Water||Total
!Sample name||Template||10xbuffer||100xbuffer||EcoRI||PstI||Water||Total
Line 1,258: Line 1,258:
|}
|}
(13:25-14:30)
(13:25-14:30)
-
====Purification====
+
===Purification====
{|class="experiments"
{|class="experiments"
|rSRRz||6.5 (ng/&micro;L)
|rSRRz||6.5 (ng/&micro;L)
Line 1,264: Line 1,264:
|low||16.8
|low||16.8
|}
|}
-
====Ligation and transformation====
+
===Ligation and transformation====
Insert: rSRRz 1-1 Vector: low copy plasmid
Insert: rSRRz 1-1 Vector: low copy plasmid
-
===Thursday, September 2 <span class="by">By: Tomonori, Tomo, Takuya, Ken<span>===
+
==Thursday, September 2 <span class="by">By: Tomonori, Tomo, Takuya, Ken<span>===
-
====Making culture and Master plate====
+
===Making culture and Master plate====
{|class="experiments"
{|class="experiments"
|rSRRz low||13 colonies
|rSRRz low||13 colonies
Line 1,275: Line 1,275:
|rSRRz low (Control)||13colonies
|rSRRz low (Control)||13colonies
|}
|}
-
====Screening PCR of rSRRz low====
+
===Screening PCR of rSRRz low====
Sample: rSRRz (1-13)  
Sample: rSRRz (1-13)  
Maker: lambda, 100
Maker: lambda, 100
Line 1,285: Line 1,285:
-
===Friday, September 3 <span class="by">By: Tomonori, Tomo, Kazuya, Tasuku, Fumitaka, Ken</span>===
+
==Friday, September 3 <span class="by">By: Tomonori, Tomo, Kazuya, Tasuku, Fumitaka, Ken</span>===
-
====Making culture====
+
===Making culture====
lac rr&Delta;TMD1 1, 3
lac rr&Delta;TMD1 1, 3
rr&Delta;TMD1 1-1, 1-2
rr&Delta;TMD1 1-1, 1-2

Revision as of 09:13, 17 October 2010

Contents

Notebook: Construction for Lysisbox

Tuesday, July 20 By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto=

Transformation=

NameWellSampleCompetent CellsTotalPlateIncubationResult
<partinfo>J23100</partinfo>1-18-C1 µl2021LB (Amp+)At 37℃, 7/20 20:50 - 7/21 17:00
<partinfo>J23105</partinfo>1-18-M12021
<partinfo>J23116</partinfo>1-20-M12021
<partinfo>R0011</partinfo>1-6-G12021
<partinfo>E0840</partinfo>1-12-O12021
<partinfo>J06702</partinfo>2-8-E12021
<partinfo>pSB4K5</partinfo>1-5-G12021×
<partinfo>B0015</partinfo>1-23-L12021LB (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 By: Wataru, Ken, Makoto, Takuya Y.=

Culture at 37℃ from 07/21 20:50 to 07/22 17:00 and Making Master Plate=

Transformation=

NameWellSampleCompetent CellsTotalPlateIncubationResult
<partinfo>pSB4K5</partinfo>1-5-G1 µl2021LB (Kan+)At 37℃, 7/21 20:50 - 7/22 16:30
<partinfo>B0015</partinfo>1-23-L12021

PCR for SRRz and S=

No.WaterMgSO4dNTPs10xBufferTemplate DNAPrimer Fwd.Primer Rev. (SRRz)Primer Rev. (S)KOD Plus ver.2Total
128 µl35551.51.5-150
22835551.51.5-150
32835551.5-1.5150
42835551.5-1.5150
52835551.51.5-150
62835551.51.5-150
72835551.5-1.5150
82835551.5-1.5150
94℃2min
98℃10sec30 cycles
55℃30sec
68℃4min
4℃forever


Thursday, July 22 By: Wataru=

Electrophoresis (40min) of the PCR Products=

KyotoExp100722-1.png
No.NameLength(bp)Result
1SRRz1386
2SRRz1386
3S442
4S442
5SRRz1386
6SRRz1386
7S442
8S442

Marker: 100bp, 1kb, 1kb, 100bp.

Miniprep=

NameConcentration
<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 By: Wataru, Tomo, Makoto=

Miniprep=

NameConcentration
<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.

PCR Purification=

No.NameConcentrationNew Name
1SRRz18.6 ng/µl-
3S77.6SSam7(1)
5SRRz33.6-
7S65.4SSam7(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.

Standard PCR for SRRz=

No.WaterMgSO4dNTPs10xBufferTemplate DNAPrimer Fwd. (SRRz)Primer Rev. (SRRz)KOD plus ver.2Total
128 µl35551.51.5150
22835551.51.5150
326.54.55551.51.5150
426.54.55551.51.5150
52565551.51.5150
62565551.51.5150
94℃2min
98℃10sec30 cycles
55℃30sec
68℃4min
4℃forever

Restriction Digestion and Electrophoresis (35min) to check function of our Restriction Enzyme=

No.NameSample10xBufferBSAEnzymeMilliQTotalIncubation
1<partinfo>J06702</partinfo>5 µl10.1EcoRI0.13.610At 37℃ 7/23 18:00 - 7/23 18:30
2<partinfo>J06702</partinfo>510.1XbaI0.13.610
3<partinfo>J06702</partinfo>510.1SpeI0.13.610
4<partinfo>J06702</partinfo>510.1PstI0.13.610
5<partinfo>J06702</partinfo>510.1-3.710
KyotoExp100723-1.png

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.

Restriction Digestion and Ligation to insert S gene to <partinfo>E0840</partinfo>=

NameSample10xBufferEnzyme 1Enzyme 2MilliQTotalIncubation
SSam7(1)11 µl5EcoRI0.2SpeI0.233.650At 37℃ for 2h
SSam7(2)115EcoRI0.2SpeI0.233.650
<partinfo>E0840</partinfo>455EcoRI0.2XbaI0.2050

After PCR Purification, evaporated them and diluted 3ul.

NameVectorInsertLigation HighTotal
SSam7(1)-E0840<partinfo>E0840</partinfo>0.5µlSSam7(1)0.512
SSam7(2)-E0840<partinfo>E0840</partinfo>0.5SSam7(2)0.512


Monday, July 26 By: Wataru, Tomonori, Makoto=

Electrophoresis of PCR Products=

KyotoExp100726-1.png
No.NameLength(bp)Result
1SRRz1386
2SRRz1386
3SRRz1386
4SRRz1386
5SRRz1386
6SRRz1386

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=

No.NameConcentrationNew Name
4SRRZ51.6 ng/µlSRRzSam7(1)
5SRRZ59.3
6SRRZ59.6SRRzSam7(2)

Transformation=

NameWellSampleCompetent CellTotalPlateIncubationResult
<partinfo>E0240</partinfo>1-12-M1 µl2021LB (Amp+)At 37℃ 7/26 - 7/27×
<partinfo>I20260</partinfo>2-17-F12021LB (Kan+)×
<partinfo>J04450</partinfo>1-5-E12021×

Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>=

Tuesday, July 27 By: Wataru, Tomo, Kazuya, Ken, Naoi=

Colony PCR of SSam7-E0840 (Electrophoresis for 35min)=

KyotoExp100727-1.png
No.NameLengthResult
1SSam7(1)-E08401522
2SSam7(1)-E08401522×
3SSam7(1)-E08401522
4SSam7(1)-E08401522×
5SSam7(1)-E08401522
6SSam7(1)-E08401522◎ (Use as SSam7(1)-E0840)
7SSam7(2)-E08401522×
8SSam7(2)-E08401522×
9SSam7(2)-E08401522×
10SSam7(2)-E08401522×
11SSam7(2)-E08401522◎ (Use as SSam7(2)-E0840)
12SSam7(2)-E08401522
13SSam7(2)-E08401522
+<partinfo>E0840</partinfo>1116
-None

Marker: 1kb, 100bp

Miniprep=

NameConcentration
<partinfo>R0011</partinfo>26.9 ng/µl
<partinfo>B0015</partinfo>120.0
<partinfo>E0840</partinfo>120.1

Restriction Digestion=

Sample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotalIncubation
<partinfo>B0015</partinfo>30 µl50.5EcoRI0.4XbaI0.313.750At 37℃ 16:45 - 18:00
SRRzSam7(1)4050.5EcoRI0.4SpeI0.43.850
SRRzSam7(2)4050.5EcoRI0.4SpeI0.43.850

Ligation=

Transformation=

NameSampleCompetent CellsTotalPlateIncubationResult
SRRzSam7(1)-B0015
SRRzSam7(2)-B0015


Wednesday, July 28 By: =

Miniprep=

NameConcentration
SSam7(1)-E084095.5 ng/µl
SSam7(2)-E084098.6

Diluted SSam7(1)-E0840 and SSam7(2)-E0840 20 times with water, and used as template DNA.

Deletion PCR to delete a functional domain of S gene=

WaterMgSO4dNTPs10xBufferPrimer Fwd.Primer Rev.SSam7(1)-E0840SSam7(2)-E0840KOD Plus ver.2Total
SSam7,ΔTMD1(1)-E0840 (1)283551.51.55-150
SSam7,ΔTMD1(1)-E0840 (2)283551.51.55-150
SSam7,ΔTMD1(2)-E0840 (1)283551.51.5-5150
SSam7,ΔTMD1(2)-E0840 (2)283551.51.5-5150
94℃2min
98℃10sec35 cycles
55℃30sec
68℃4min
4℃forever

Restriction Digestion to check the function of DpnI=

NameSamplefast digestion bufferDpnIMilliQTotal
SSam7,ΔTMD1(1)-E0840 (1)310.15.810
SSam7,ΔTMD1(2)-E0840 (2)310.15.810

Electrophoresis for 35min=

KyotoExp100728-1.png
No.NameLengthResult
1Not digested SSam7(1)-E08403363
2Not digested SSam7(2)-E08403363
3Digested SSam7(1)-E08401021, 933, 402, 341, 258, 105, ...
4Digested SSam7(2)-E08401021, 933, 402, 341, 258, 105, ...

Marker: 1kb, 100bp DpnI works correctly.


Thursday, July 29 By: =

Restriction Digestion=

NameSample volumeFastdigestion BufferEnzyme 1MilliQTotalIncubation
SSam7,ΔTMD1(1)-E0840 (1)50 µl6DpnI0.23.86007/29 09:40 - 07/29 11:00
SSam7,ΔTMD1(2)-E0840 (1)506DpnI0.23.860

Ligation and Phosphorylation=

NameSampleMilliQLigation HighT4 KinaseTotalIncubation
SSam7,ΔTMD1(1)-E0840 (1)2 µl7511507/29 11:30 ~ 07/29 13:00
SSam7,ΔTMD1(1)-E0840 (2)275115

Transformation=

NameSample VolumeCompetent CellTotalPlateIncubationResult
SSam7,ΔTMD1(1)-E0840 (1)3 µl3033LB Amp+07/29 ~ 07/30
SSam7,ΔTMD1(1)-E0840 (2)33033


Monday, August 2 By: Wataru, Ken=

Miniprep=

NameConcentration(ng/µL)
SΔTMD1-E0840-152.7
SΔTMD1-E0840-254.4
SΔTMD1-E0840-389.5
<partinfo>pSB4K5</partinfo>50.7
<partinfo>R0011</partinfo>18.6

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.

NameWater25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2Primer VF2(10µM)Primer VR(10µM)Template E240KOD Pllus ver.2Total
E02401283551.51.55150
E02402283551.51.55150
94℃2min
98℃10sec35 cycles
55℃30sec
68℃4min
4℃forever

Electrophoresis=

PCR Purification=

Sample numberConcentration(ng/µL)
E0240142.6
E0240255.3

Restriction Digestion for inserting <partinfo>E0240</partinfo> to pSB4K5 by 3A assembly=

NameSample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
E02401(X-P)3050.5XbaI0.2PstI0.214.150
E02402(X-P)3050.5XbaI0.2PstI0.214.150

PCR Purification=

NameConcentration(ng/µL)Volume(µL)
E02401(X-P)21.840
E02402(X-P)32.445

Stored at -20℃.

Error PCR=

NameWater25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2Primer VF2(10µM)Primer VR(10µM)Template Δ1TemplateTemplateKOD Pllus ver.2Total
SΔTMD1-E08401-1323551.51.51--150
SΔTMD1-E08401-2323551.51.5-1-150
SΔTMD1-E08402323551.51.5--1150
94℃2min
98℃10sec20 cycles
68℃4min
4℃forever

Transformation=

NameSample (µl)Competent Cells (µl)Total (µl)PlateIncubationResult
SΔTMD1-E08401-122022rowspan="3"rowspan="3"
SΔTMD1-E08401-222022}
SΔTMD1-E0840222022


Tuesday, August 3 By: =

Culture of each two colonies of SΔTMD1-E08401-1 and SΔTMD1-E08402 for 37℃ 08/03-08/04=

Miniprep for Construction of Measure(lacP) and Measure(Standard)=

Sample numberConcentration(ng/µL)
<partinfo>pSB4K5</partinfo>60.7
<partinfo>R0011</partinfo>26.8

Restriction Digestion=

NameSample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
R00115060.6EcoRI0.2SpeI0.2360
pSB4K5(E-P)5060.6EcoRI0.2PstI0.2360
E02401(X-P)5060.6XbaI0.2PstI0.2360
E02402(X-P)5060.6XbaI0.2PstI0.2360

PCR Purification=

Sample numberConcentration(ng/µL)
pSB4K5(E-P)39.5
E02401(X-P)21.8
E02402(X-P)32.4

pSB4K5(E-P) is concentrated 10µL and E02401(X-P), E02402(X-P) are concentrated 1µL.

Ethanol Precipitation=

Dilution of <partinfo>pSB4K5</partinfo> by 2µl MilliQ=

Ligation=

VectorInsert 1Insert 2Ligation HighTotalIncubation
R0011-E02401[Low]pSB4K5(E-P)1R0011(E-S)1E02401(X-P)131517:30 - 20:20
R0011-E02402[Low]pSB4K5(E-P)1R0011(E-S)1E02402(X-P)1315

Standard PCR of <partinfo>J23101</partinfo>-<partinfo>E0240</partinfo> that is important in the measurement of RPU=

NameWater25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2Primer VF2(10µM)Primer VR(10µM)Template J23101-E0240KOD plus ver.2 Total
J23101-E02401323551.51.51150
J23101-E02402323551.51.5-150
94℃2min
98℃10sec30 cycles
55℃30sec
68℃4min
4℃forever

PCR Purification=

NameConcentration(ng/µL)
J23101-E024040.6

Restriction Digestion=

NameSample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
J23101-E0240(E-P)4560.6EcoRI0.2PstI0.2860

PCR Purification=

NameConcentration(ng/µL)Volume(µL)
J23101-E0240(E-P)74.130

J23101-E0240(E-P) is concentrated 7µL

Ligation=

VectorInsertLigation HighTotalIncubation
J23101-E0240[Low]pSB4K5(E-P)1J23101-E0240(E-P)12420:00-20:30

Transformation=

NameConc(/µL)Sample Volume(µL)Competent Cell(µL)TotalPlateIncubation
R0011-E02401[Low]-12021LB kan8/3~8/4
R0011-E02402[Low]-12021
J23101-E0240[Low]-12021


Thursday, August 5 By: =

Result of Transformation=

R0011-E02401[Low]Many colonies
R0011-E02402[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-E02401[Low] and R0011-E02402[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.

NameColorIncubation
R0011-E02401[Low]-1Green Colony8/5-8/6
R0011-E02401[Low]-2Green Colony
R0011-E02401[Low]-3White Colony
R0011-E02401[Low]-4White Colony
R0011-E02402[Low]-1Green Colony
R0011-E02402[Low]-2White Colony
R0011-E02402[Low]-3White Colony
R0011-E02402[Low]-4White Colony
J23101-E0240[Low]-1Green Colony
J23101-E0240[Low]-2Green Colony
J23101-E0240[Low]-3Green Colony
NameConcentration(ng/µL)
SΔTMD1-E08401-1-A28.9
SΔTMD1-E08401-1-B25.3
SΔTMD1-E08402-A26.6
SΔTMD1-E08402-B24.0

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=

Name
R0011-E02401[Low]-1
R0011-E02401[Low]-2
R0011-E02401[Low]-3
R0011-E02401[Low]-4
R0011-E02402[Low]-1
R0011-E02402[Low]-2
R0011-E02402[Low]-3
R0011-E02402[Low]-4
J23101-E0240[Low]-1
J23101-E0240[Low]-2
J23101-E0240[Low]-3

Restriction Digestion=

NameSample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
R0011-E02401[Low]-15060.6EcoRI0.3PstI0.32.860
R0011-E02401[Low]-25060.6EcoRI0.3PstI0.32.860
R0011-E02401[Low]-35060.6EcoRI0.3PstI0.32.860
R0011-E02401[Low]-45060.6EcoRI0.3PstI0.32.860
R0011-E02402[Low]-15060.6EcoRI0.3PstI0.32.860
R0011-E02402[Low]-25060.6EcoRI0.3PstI0.32.860
R0011-E02402[Low]-35060.6EcoRI0.3PstI0.32.860
R0011-E02402[Low]-45060.6EcoRI0.3PstI0.32.860
J23101-E0240[Low]-15060.6EcoRI0.3PstI0.32.860
J23101-E0240[Low]-25060.6EcoRI0.3PstI0.32.860
J23101-E0240[Low]-35060.6EcoRI0.3PstI0.32.860
Electrophoresis
M100bpcolspan="4"colspan="4"|
Mλ
Mλ
M100bp
1J23101-E0240[Low]-1
2J23101-E0240[Low]-2
3J23101-E0240[Low]-1
4R0011-E02401[Low]-1
5R0011-E02401[Low]-2
6R0011-E02401[Low]-3}
7R0011-E02401[Low]-4}
8R0011-E02402[Low]-1
9R0011-E02402[Low]-2}
10R0011-E02402[Low]-3}
11R0011-E02402[Low]-4}
12J23101-E0240[Low]-1
13J23101-E0240[Low]-2

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)=

NameWater25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2Primer VF2(10µM)Primer VR(10µM)Template ΔTMD failed(50ng/µL)KOD plus ver.2Total
ΔTMD①323551.51.51150
ΔTMD②323551.51.51150
94℃2min
98℃10sec25 cycles
68℃4min
Add DpnI 2µl
Incubate1h
4℃forever

Transformation=

NameConc(/µL)Sample Volum(µL)Competent Cell(µL)TotalPlateIncubation
ΔTMD①-45054LB kan8/6~8/9
ΔTMD②-45054
2-17-F-25052
2-I-525052LB amp


Monday, August 9 By: Wataru, Tomonori, Ken, Takuya=

Miniprep of MS and ML=

Sample numberconcentration(ng/µL)
MS116.2
ML146.6

Transfotrmation of MS and ML=

Sampleconc(ng/µL)Sample vol(µL)Competent CellCompetent cell vol(µL)Total vol(µL)PlateIncuvation
MS116.22KRX5052LB kanamycin8/9 18:00‾8/10 12:00
ML146.62KRX5052

Restriction enzyme digestion and ethanol precipitation=

To use lac p for next ligation, we digested 1-6-G by EroRI and PstI

Sample10x BufferBSAEnzyme (EcoRI)Enzyme (PstI)MilliQTotal
5060.60.50.52.460

Incubate 37℃ 8/9 16:20‾18:20 After restriction enzyme digestion, we did ethanol precipitation.

Ligation and Transformation=

SampleConc (nu/µL)Sample vol (µL)Competent cellCompetent cell vol (µL)Total vol (µL)PlateIncuvation
Lac p (low)-2KRX5052LB kanamycin8/9 20:00‾8/10 9:00
2C25052


==Tuesday, August 10 By: Wataru, Tomonori, Ken, Fumitaka

Making culture plate on lac p (low), MS and ML=

Lac p (low)KRXMany colonies
C2
MSKRX
MLKRX

Minprep of ΔTMD1+GFP=

Sample numberConcentration (ng/µL)
1-19.9
1-227.3
2-143.2
2-234.7

37℃ 8/9 18:00‾8/10 9:00

Culture and Master Plate=

==Wednesday, August 11 By: Wataru, Naoi, Ken, Takuya

SampleMediumCloudIncubation
1Kanamycino37℃8/10 20:00‾8/11 9:00
Ampicillinx
2Kanamycino
Ampicillino
3Kanamycino
Ampicillinx
4Kanamycino
Ampicillinx
5Kanamycino
Ampicillinx
6Kanamycino
Ampicillino
7Kanamycino
Ampicillinx

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=

NameEcoRIPstI
10.2-
2-0.2
30.20.2
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 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 KyotoExp100811-2.png 7 8 9 10 11 12 13 M 14 15 16 18 19 20 M KyotoExp100811-3.png Discussion: All of the sample were self-ligation of DT and SRRz weren't inserted.


Thursday, August 12 By: Wataru, Ken=

Restriction Digestion and electrophoresis of <partinfo>B0015</partinfo>=

Sample nameTemplate10xbuffer100xbufferEcoRIXbaI 1XbaI 2SpeIPstI 1PstI 2WaterTotal
1310.10.2-----5.710
2310.1-0.2----5.710
3310.1--0.2---5.710
4310.1---0.2--5.710
5310.1----0.2-5.710
6310.1-----0.25.710
N310.1------5.910

Sample: 1-6, N Maker: lambda, 100 M 1 2 3 4 5 6 N M M M KyotoExp100812-1.png Discussion: Each enzyme correctly cut each sample and was active.


==Thursday, August 19 By: Wataru, Tomo, Ken

Miniprep of SΔTMD1GFP=

29.6(ng/µg)

Point mutation PCR of ΔTMD1GFP=

Sample numberTemplate10xbufferdNTPsMgSO4Primer 1Primer 2WaterKOD-plus-Total
11.55531.51.531.5150
21.55531.51.531.5150
control1.55531.51.532.5-50
94(℃)2min
9810sec30cycles
5530sec
683.5min
4.0forever

Restriction Digestion(DpnI): 17:50-18:50=

Electrophoresis=

Sample: 1, 2, Control Marker: lambda, 100 KyotoExp100819-1.png

Ligation and Transformation=

We named point mutation PCR products rΔTMD1GFP.


Monday, August 23 By: Wataru, Tomo, Ken, Fumitaka, Tasuku=

Miniprep of ΔTMD1=

Sample numberConcentration(ng/µg)
1-158.9
2-249.9

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

90℃10min
94℃30sec35cycles
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 KyotoExp100823-1.png Discussion: We found the band; about 200bp, and it meant the lligation was completed successfully.

Deletion PCR of rΔTMD1GFP 2-2=

Sample10xdNTPsPrimer1Primer2TemplateWaterKOD-plus-Total
1551.51.5135150
2551.51.5135150
Control551.51.5135-50
94℃2min
94℃10sec35cycles
56℃30sec
68℃3.5min
4℃hold

Restriction Digestion(DpnI)=

Template25(µL)
DpnI1
Total26

19:10-20:10

Ligation=

SampleTemplateWaterLigation highT4 Kinasetotal
1365115
2365115
Control365115

20:15-21:15

Transformation=

We named sample 1, 2 and control rrδTMD1GFP1, 2 and control.


Tuesday, August 24 By:Ken, Tomo, Tasuku, Takuya=

Retry of deletion PCR of rδTMD1 GFP=

Sample10xdNTPsMgSO4Primer1Primer2TemplateWaterKOD-plus-Total
15531.51.5132150
25531.51.5132150
Control5531.51.5132150
94℃2min
94℃10sec35cycles
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 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=

Sample10xdNTPsMgSO4Primer1Primer2TemplateWaterKOD-plus-total
15531.51.5132150
25531.51.5132150
control5531.51.5132150
94℃2min
98℃10sec30cycles
55℃30sec
68℃4min
4℃hold

Restriction Digestion(DpnI), electrophoresis and ligation=

KyotoExp100824-2.png We could find point mutation PCR and restriction enzyme of DpnI was done.


PCR of E0240=

Sample10}dNTPsMgSO4VF2VRTemplateWaterKOD-plus-Total
15531.51.5131.5150
25531.51.5131.5150


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 By:Ken, Tomo, Kazuya, Tasuku, Takuya=

Making culture and Master plate=

rrΔTMD1-1Many Colonies
rrΔTMD1-2
rrΔTMD1-C-zero
rSRRz-1Many 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=

Sample nameTemplate10xbuffer100xbufferEcoRISpeIPstIWaterTotal
1-5G5060.60.40.4-2.660
Lac low1040.4-0.30.32540
Sample NameConcentration(ng/µL)
1-5G18.4
Lac low8.6

Ligation of <partinfo>E0240</partinfo> and <partinfo>pSB4K5</partinfo>, Transformation=

==Thursday, August 26 By:Ken, Tomo, Kazuya, Tasuku, Takuya, Fumitaka

Miniprep=

Sample nameConcentration(ng/µL)
constP(0.7)44.5

Restriction Digestion of constP(0.7)=

Template10xbuffer100xbufferSpeIPstIWaterTotal
2540.40.30.31040

Purification of constP (0.7)=

49.8 ng/µL


Friday, August 27 By:Ken, Tomo, Kazuya, Fumitaka=

Making master plate of E0240 low=

Sample NameConcentration(ng/µL)
rrΔTMD1 1-220.9
rSRRz 1-116.4

Restriction Digestion of rrΔTMD1 and rSRRz=

Sample nameTemplate10xbuffer100xbufferXbaIPstIWaterTotal
rrΔTMD1 1-24560.60.30.37.860
rSRRz 1-14560.60.30.37.860

(13:20-14:20)

Purification=

rrΔTMD1 1-244.7
rSRRz 1-156.1

Lagation and transformation=

lacP + rrΔTMD1 1-2 constP (0.7) + rrΔTMD1 1-2 lac low + rSRRz 1-1


Monday, August 30 By: Tomonori, Kazuya, Tasuku, Ken=

Making culture and Master plate=

lacP rrΔTMD1GFPMany colonies
lacP rrΔTMD1GFP(control)Some colonies
constP rrΔTMD1GFPMany colonies
constP rrΔTMD1GFP(control)Many colonies
lacP rSRRz lowNo 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 By: Tomonori, Takuya Y., Kazuya, Tasuku, Takuya, Ken

Miniprep=

constP (0.3)48.5 (ng/µL)
lac rrΔTMD1107.3

RE of constP (0.3) and lac rrΔTMD1=

Gel Extraction of lac rrΔTMD1=

File: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=

constP (0.3)5.8 (ng/µL)
lac rrΔTMD17.8 (ng/µL)

Ligation and transformation=

InsertVector
lac rrΔTMD1constP (0.3)


==Wednesday, September 1 By: Tomonori, Kazuya, Tasuku, Fumitaka, Ken

Making culture and Master plate=

lac rrΔTMD1 constPmany 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

File:KyotoExp100901.png

Discussion: All of the sample except sample 10 might be self-ligation products of constP.

Miniprep=

rSRRz 1-133.8 (ng/µL)
low56.0 (ng/µL)

Restriction Digestion of rSRRz and low=

Sample nameTemplate10xbuffer100xbufferEcoRIPstIWaterTotal
rSRRz2040.40.30.31540
low2040.40.30.31540

(13:25-14:30)

Purification=

rSRRz6.5 (ng/µL)
low16.8

Ligation and transformation=

Insert: rSRRz 1-1 Vector: low copy plasmid


Thursday, September 2 By: Tomonori, Tomo, Takuya, Ken=

Making culture and Master plate=

rSRRz low13 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 File: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 By: Tomonori, Tomo, Kazuya, Tasuku, Fumitaka, Ken=

Making culture=

lac rrΔTMD1 1, 3 rrΔTMD1 1-1, 1-2 rSRRz 1-1, 1-2 ML