Team:Kyoto/Notebook

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==Index==
==Index==
==Notebook==
==Notebook==
-
 
+
===Tuesday, July 20 <span class="by">By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto</span>===
-
<div class="note">
+
<div class="preparation">
-
===Tuesday, July 20===
+
====Solubilization of Antibiotics====
-
By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
+
-
 
+
-
====1. Solubilization of antibiotics.====
+
-
For Ampicillin(Amp), add 1.0g Amp to 20ml MilliQ (final concentration is 50mg/ml). For Kanamycin(Kan), add 0.5g Kan to 10ml MilliQ (final concentration is 50mg/ml). Dispense 1.1ml of the solution into 1.5ml tubes and store in the freezer (-20&#x2103;).
+
-
 
+
-
====2. Make plates for LB (Amp+) and LB (Kan+).====
+
-
 
+
-
====3. [[Team:Kyoto/Protocols#Transformation|Transformation]] of iGEM Parts.====
+
{| class="experiments"
{| class="experiments"
-
!Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
+
|Ampicillin(Amp)||Kanamycin(Kan)||
|-
|-
-
|<partinfo>J23100</partinfo>||1-18-C||1||20||21||rowspan="7"|LB (Amp+)||rowspan="8"|At 37&#x2103; 7/20 20:50 - 7/21 17:00||○
+
|Mix 1.0g Amp and 20ml MilliQ||Mix 0.5g Kan and 10ml MilliQ||Final concentration is 50mg/ml
|-
|-
-
|<partinfo>J23105</partinfo>||1-18-M||1||20||21||○
+
|colspan="2"|Dispense 1.1ml of the solution into 1.5ml tubes||
|-
|-
-
|<partinfo>J23116</partinfo>||1-20-M||1||20||21||○
+
|colspan="2"|Store in the freezer (-20&#x2103;)||
 +
|}
 +
</div>
 +
----
 +
<div class="plate">
 +
====Making plates for LB (Amp+) and LB (Kan+)====
 +
</div>
 +
----
 +
<div class="transformation lysis measure">
 +
====[[Team:Kyoto/Protocols#Transformation|Transformation]]====
 +
{| class="experiments"
 +
!Name||Well||Sample (&micro;l)||Competent Cells (&micro;l)||Total (&micro;l)||Plate||Incubation||Result
|-
|-
-
|<partinfo>R0011</partinfo>||1-6-G||1||20||21||○
+
|<partinfo>J23100</partinfo>||1-18-C||1||20||21||rowspan="7"|LB (Amp+)||rowspan="8"|At 37&#x2103;, 7/20 20:50 - 7/21 17:00||&#x25CB;
|-
|-
-
|<partinfo>E0840</partinfo>||1-12-O||1||20||21||○
+
|<partinfo>J23105</partinfo>||1-18-M||1||20||21||&#x25CB;
|-
|-
-
|<partinfo>J06702</partinfo>||2-8-E||1||20||21||○
+
|<partinfo>J23116</partinfo>||1-20-M||1||20||21||&#x25CB;
|-
|-
-
|<partinfo>pSB4K5</partinfo>||1-5-G||1||20||21||×
+
|<partinfo>R0011</partinfo>||1-6-G||1||20||21||&#x25CB;
|-
|-
-
|<partinfo>B0015</partinfo>||1-23-L||1||20||21||LB (Kan+)||×
+
|<partinfo>E0840</partinfo>||1-12-O||1||20||21||&#x25CB;
 +
|-
 +
|<partinfo>J06702</partinfo>||2-8-E||1||20||21||&#x25CB;
 +
|-
 +
|<partinfo>pSB4K5</partinfo>||1-5-G||1||20||21||&#x25CF;
 +
|-
 +
|<partinfo>B0015</partinfo>||1-23-L||1||20||21||LB (Kan+)||&#x25CF;
|}
|}
A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Amp+). And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance. So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".
A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Amp+). And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance. So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".
</div>
</div>
-
 
+
===Wednesday, July 21 <span class="by">By: Wataru, Ken, Makoto, Takuya Y.</span>===
-
<div class="note">
+
<div class="culture lysis measure">
-
===Wednesday, July 21===
+
====Culture of plates in which colonies was observed at 37&#x2103; from 07/21 20:50 to 07/22 17:00====
-
By: Wataru, Ken, Makoto, Takuya Yamamoto
+
</div>
-
 
+
----
-
====1. Culture plates in which colonies was observed at 37&#x2103; from 07/21 20:50 to 07/22 17:00.====
+
<div class="master lysis measure">
-
 
+
====Making a master plate of the above plates====
-
====2. Make a master plate of the above plates.====
+
</div>
-
 
+
----
-
====3. Retry [[Team:Kyoto/Protocols#Transformation|Transformation]] of iGEM Parts.====
+
<div class="transformation lysis measure">
-
{| class="experiments"
+
====Retry [[Team:Kyoto/Protocols#Transformation|Transformation]]===
!Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
!Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
|-
|-
Line 51: Line 60:
|<partinfo>B0015</partinfo>||1-23-L||1||20||21||○
|<partinfo>B0015</partinfo>||1-23-L||1||20||21||○
|}
|}
-
 
+
</div>
-
====4. [[Team:Kyoto/Protocols#PCR|PCR]] for S-R-Rz/Rz1 and S====
+
----
-
Dilute &lambda;DNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template &lambda;DNA was 5ng/µl.
+
<div class="pcr lysis">
 +
====[[Team:Kyoto/Protocols#Stantard_PCR|PCR]] for S-R-Rz/Rz1 and S====
{| class="experiments"
{| class="experiments"
!No.||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||TemplateDNA (5ng/µl)||Primer Forward (10µM)||Primer S-R-Rz/Rz1 Reverse (10µM)||Primer S Reverse (10µM)||KOD Plus ver.2||Total
!No.||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||TemplateDNA (5ng/µl)||Primer Forward (10µM)||Primer S-R-Rz/Rz1 Reverse (10µM)||Primer S Reverse (10µM)||KOD Plus ver.2||Total
Line 73: Line 83:
|8||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
|8||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
|}
|}
-
Forward Primer of S-R-Rz/Rz1 and S is common. PCR condition: 94&#x2103; x 2min, (98&#x2103; x 10sec, 55&#x2103; x 30sec, 68&#x2103; x 1min) x 30cycles, 4&#x2103; forever.
 
-
</div>
 
-
 
-
<div class="note">
 
-
===Thursday, July 22===
 
-
By: Wataru
 
-
 
-
====1. [[Team:Kyoto/Protocols#Electrophoresis|Electrophoresis]] of the PCR products for 40min.====
 
-
[[Image:KyotoExp100722-1.png|300px|right]]
 
-
Length of S and S-R-Rz/Rz1 is 370bp, 1300bp, so PCR succeeded.
 
-
 
-
====2. [[Team:Kyoto/Protocols#Miniprep|Miniprep]] of iGEM Parts.====
 
-
{| class="experiments"
 
-
!Name||Concentration(ng/&micro;l)
 
-
|-
 
-
|<partinfo>J23100</partinfo>||18.5
 
-
|-
 
-
|<partinfo>J23105</partinfo>||12.5
 
-
|-
 
-
|<partinfo>J23116</partinfo>||14.6
 
-
|-
 
-
|<partinfo>R0011</partinfo>||8.6
 
-
|-
 
-
|<partinfo>E0840</partinfo>||12.1
 
-
|-
 
-
|<partinfo>J06702</partinfo>||14.7
 
-
|}
 
-
The concentration of all samples was very week. Probably our shaking incubation was week.
 
-
 
-
====3. Culture plates and make master plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo> from 7/22 17:00 to 7/23 10:00.====
 
-
</div>
 
-
 
-
<div class="note">
 
-
===Friday, July 23===
 
-
By: Wataru, Tomo, Makoto
 
-
 
-
====1. [[Team:Kyoto/Protocols#Miniprep|Miniprep]] of iGEM Parts.====
 
-
{| class="experiments"
 
-
!Name||Concentration(ng/&micro;l)
 
-
|-
 
-
|<partinfo>pSB4K5</partinfo>||79.2
 
-
|-
 
-
|<partinfo>B0015</partinfo>||-
 
-
|}
 
-
We lost <partinfo>B0015</partinfo> by our mistake. The concentration of <partinfo>pSB4K5</partinfo> is high, so this condition of shaking incubation is moderate.
 
-
 
-
====2. Picked up 1, 3, 5, 7 of the products of PCR, and purified by PCR-purification.====
 
-
{| class="experiments"
 
-
!Sample||Concentration (ng/&micro;l)||New Name
 
-
|-
 
-
|1||18.6||-
 
-
|-
 
-
|3||77.6||S<sub>1</sub>
 
-
|-
 
-
|5||33.6||-
 
-
|-
 
-
|7||65.4||S<sub>2</sub>
 
-
|}
 
-
The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.
 
-
 
-
====3. Retry of PCR of S-R-Rz/Rz1.====
 
-
{| class="experiments"
 
-
!Sample||Water||25mmol/l MgSO4||2mmol/l dNTPs||10×Buffer for KOD plus ver.2||Template DNA (5ng/&micro;l)||Primer S-R-Rz/Rz1 Forward (10&micro;mol/l)||Primer S-R-Rz/Rz1 Reverse (10&micro;mol/l)||KOD plus ver.2||Total
 
-
|-
 
-
|1||28&micro;l||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
 
-
|}
 
-
PCR condition : 94&#x2103; x 2min, (98&#x2103; x 10sec, 55&#x2103; x 30sec, 68&#x2103; x 1min) x 30cycles, 4&#x2103; forever.
 
-
 
-
====4. Digested <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, PstI to check function of our Restriction enzymes.====
 
-
{| class="experiments"
 
-
!Sample||10xBuffer||BSA||Enzyme||MilliQ||Total||Incubation
 
-
|-
 
-
|1||5&micro;l||1||''EcoR''I 0.1||3.6||10||rowspan="5"|At 37&#x2103; 7/23 18:00 - 7/23 18:30
 
-
|-
 
-
|2||5||1||''Xba''I 0.1||3.6||10
 
-
|-
 
-
|3||5||1||''Spe''I 0.1||3.6||10
 
-
|-
 
-
|4||5||1||''Pst''I 0.1||3.6||10
 
-
|-
 
-
|5||5||1||-||3.7||10
 
-
|}
 
-
 
-
====5. Electrophoresis of above sample for 35min.====
 
-
[[Image:KyotoExp100723-1.png|300px|right]]
 
-
Comparison to sample 5(control, circular DNA), the bands of sample 1, 2, 3, 4 was shifted. The DNA of sample 1, 2, 3, 4 was linearized by Restriction enzymes.  So, our restriction enzymes work correctly.
 
-
 
-
====6. To insert S gene to GFP, we digested the PCR products of S gene by EcoRi and SpeI, and GFP by EcoRl and XbaI.====
 
-
{| class="experiments"
 
-
!Sample||10×Buffer||Enzyme 1||Enzyme 2||MilliQ||Total||Incubation
 
-
|-
 
-
|S<sub>1</sub>||11&micro;l||5||''EcoR''I 0.2||''Spe''I 0.2||33.6||50||rowspan="3"|At 37&#x2103; for 2h
 
-
|-
 
-
|S<sub>2</sub>||11||5||''EcoR''I 0.2||''Spe''I 0.2||33.6||50
 
-
|-
 
-
|<partinfo>E0840</partinfo>(GFP)||45||5||''EcoR''I 0.2||''Xba''I 0.2||0||50
 
-
|}
 
-
After PCR purification, evaporated them and diluted 3ul.
 
-
 
-
====7. Ligated over night.====
 
-
{| class="experiments"
 
-
!Sample||Vector||Insert||Ligation High||Total
 
-
|-
 
-
|S-E0840<sub>1</sub>||<partinfo>E0840</partinfo> 0.5&micro;l||S<sub>1</sub> 0.5||1||2
 
-
|-
 
-
|S-E0840<sub>2</sub>||<partinfo>E0840</partinfo> 0.5||S<sub>2</sub> 0.5||1||2
 
-
|}
 
-
</div>
 
-
 
-
<div class="note">
 
-
 
-
===Monday, July 26===
 
-
By: Wataru, Tomonori, Makoto
 
-
 
-
====1. Electrophoresis of PCR products====
 
-
[[Image:KyotoExp100726-1.png|300px|right]]
 
-
At the condition 4 (4.5&micro;l MgSO4) and 6 (6&micro;l MgSO4), S-R-Rz/Rz1 is amplified very much. So we decided to use them.
 
-
 
-
====2. PCR purification====
 
-
{| class="experiments"
 
-
!Sample||Concentration (ng/&micro;l)||New Name
 
-
|-
 
-
|4||51.6||SRRz<sub>1</sub>
 
-
|-
 
-
|5||59.3||
 
-
|-
 
-
|6||59.6||SRRz<sub>2</sub>
 
-
|}
 
-
 
-
====3. Transformation of iGEM Parts====
 
-
{| class="experiments"
 
-
!Name||Well||Sample (&micro;l)||Competent Cell (&micro;l)||Total (&micro;l)||Plate||Incubation||Result
 
-
|-
 
-
|||1-12-M||1||20||21||LB (Amplicillin+)||rowspan="3"|At 37&#x2103; 7/26 - 7/27||×
 
-
|-
 
-
|||2-17-F||1||20||21||rowspan="2"|LB (Kanamycin+)||×
 
-
|-
 
-
|||1-5-E||1||20||21||×
 
-
|}
 
-
 
-
====4. Culture of 1-6-G, 1-12-O, and 1-23-L====
 
-
</div>
 
-
 
-
<div class="note">
 
-
===Tuesday, July 27===
 
-
By: Wataru, Tomo, Kazuya, Ken, Naoi
 
-
 
-
====1. Colony PCR of S-E840====
 
-
To check that S GFP is correctly inserted, we did colony PCR. Electrophoresis was done for 35min.
 
{|class="experiments"
{|class="experiments"
-
|Marker||1||2||3||4||5||6||7||8||9||10||11||12||13||+||-||Marker
+
|+ PCR condition
|-
|-
-
|1kb||colspan="6"|S-E0840<sub>1</sub>||colspan="7"|S-E0840<sub>2</sub>||E0840||None||100bp
 
-
|}
 
-
 
-
[[image:KyotoExp100727-1.png|300px|right]]
 
-
As a result, 1,3,5,6,11,12,13 are inserted S gene correctly. So, we decided to use 6 as S-E0840<sub>1</sub>　and 11 as S-E0840<sub>2</sub>.
 
-
 
-
====2. Miniprep====
 
-
{|class="experiments"
 
-
!Sample number||Concentration(ng/&micro;L)
 
-
|-
 
-
|1-6-G||26.9
 
-
|-
 
-
|1-23-L||120.0
 
-
|-
 
-
|1-12-O||120.1
 
-
|}
 
-
 
-
====3. Restriction Digestion ====
 
-
{|class="experiments"
 
-
!||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
 
-
|-
 
-
|1-23-L||30||5||0.5||rowspan="3"|EcoRI||0.4||rowspan="3"|XbaI||0.3||13.7||50
 
-
|-
 
-
|4.5②||40||5||0.5||0.4||0.4||3.8||50
 
-
|-
 
-
|6②||40||5||0.5||0.4||0.4||3.8||50
 
-
|}
 
-
Incubate 37&#x2103; 16:45~18:00
 
-
 
-
====4. Ligation====
 
-
 
-
====5. Transformation====
 
-
 
-
===Wednesday, July 28===
 
-
 
-
====1. Result of Transformation====
 
-
{|class="experiments"
 
-
|SRRz①-DT||rowspan="2"|Many colonies
 
-
|-
 
-
|SRRz②-DT
 
-
|}
 
-
 
-
====1. Miniprep====
 
-
{|class="experiments"
 
-
!Sample number||Concentration(ng/&micro;)
 
-
|-
 
-
|S-E0840<sub>1</sub>||95.5
 
-
|-
 
-
|S-E0840<sub>2</sub>||98.6
 
-
|}
 
-
Diluted S-E0840<sub>1</sub> and S-E0840<sub>2</sub> 20 times with water, and used as template DNA.
 
-
 
-
====2. Deletion PCR====
 
-
To delete functional domain of S gene, we did deletion PCR.
 
-
{|class="experiments"
 
-
!||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer Forward(10&micro;M)||Primer Reverse(10&micro;M)||Template S-E0840<sub>1</sub>||Template S-E0840<sub>2</sub>||KOD Plus ver.2||Total
 
-
|-
 
-
|S&Delta;1-1||28||3||5||5||1.5||1.5||5||-||1||50
 
-
|-
 
-
|S&Delta;1-2||28||3||5||5||1.5||1.5||5||-||1||50
 
-
|-
 
-
|S&Delta;2-1||28||3||5||5||1.5||1.5||-||5||1||50
 
-
|}
 
-
 
-
PCR program
 
-
{|class="experiments"
 
-
|94&#x2103;||2min||
 
-
|-
 
-
|98&#x2103;||10sec||rowspan="3"|35 cycles
 
-
|-
 
-
|55&#x2103;||30sec
 
-
|-
 
-
|-
 
-
|68&#x2103;||4min
 
-
|-
 
-
|4&#x2103;||forever||
 
-
|}
 
-
 
-
====3. Restriction Digestion====
 
-
To check function of our Restriction enzymes, we digested S-E0840<sub>1</sub> and S-E0840<sub>2</sub> by DpnI.
 
-
{|class="experiments"
 
-
!||Sample||fast digestion buffer||DpnI||MilliQ||Total
 
-
|-
 
-
|S-E840①||3||1||0.1||5.8||10
 
-
|-
 
-
|S-E840②||3||1||0.1||5.8||10
 
-
|}
 
-
 
-
====4. Electrophoresis====
 
-
Electrophorese for 35min.
 
-
{|class="experiments"
 
-
|Marker||1||2||3||4||Marker
 
-
|-
 
-
|1kb||Not digested S-E0840<sub>1</sub>||Not digested S-E0840<sub>2</sub>||Digested S-E0840<sub>1</sub>||Digested S-E0840<sub>2</sub>||100bp
 
-
|}
 
-
 
-
[[image:KyotoExp100728-1.png|300px|right]]
 
-
 
-
DpnI works correctly.
 
-
 
-
===Thursday, July 29===
 
-
 
-
====1. Restriction Digestion====
 
-
{|class="experiments"
 
-
!||Sample volume||Fastdigestion buffer||Enzyme 1||MilliQ||Total
 
-
|-
 
-
|S&Delta;1-1||50||6||DpnI 0.2||3.8||60
 
-
|-
 
-
|S&Delta;2-1||50||6||DpnI 0.2||3.8||60
 
-
|}
 
-
Incubate 7/29 9:40~7/29 11:00.
 
-
 
-
====2. Ligation and Pospholylation====
 
-
{|class="experiments"
 
-
!||Sample||MilliQ||Ligation High||T4 Kinase||Total
 
-
|-
 
-
|S&Delta;1-1||2||7||5||1||15
 
-
|-
 
-
|S&Delta;2-1||2||7||5||1||15
 
-
|}
 
-
Incubate 7/29 11:30~7/29 13:00.
 
-
 
-
====3. Transformation====
 
-
{|class="experiments"
 
-
!Sample||Conc(/&micro;L)||Sample Volume(&micro;L)||Competent Cell(&micro;L)||Total||Plate||Incubation
 
-
|-
 
-
|S&Delta;1-1||-||3||30||33||rowspan="2"|LB amp||rowspan="2"|7/29~7/30
 
-
|-
 
-
|S&Delta;1-1||-||3||30||33
 
-
|}
 
-
 
-
===Friday, July 30===
 
-
Result of transformation of &Delta;1 and &Delta;2
 
-
Many colonies are observed.
 
-
 
-
===Monday, August 2===
 
-
By: Wataru, Ken
 
-
 
-
====1. Miniprep====
 
-
 
-
{|class="experiments"
 
-
!Sample number||Concentration(ng/&micro;L)
 
-
|-
 
-
|&Delta;1||52.7
 
-
|-
 
-
|&Delta;2||54.4
 
-
|-
 
-
|&Delta;3||89.5
 
-
|-
 
-
|pSB4K5||50.7
 
-
|-
 
-
|LacP||18.6
 
-
|}
 
-
 
-
====2. PCR and RE of E240====
 
-
 
-
E240 is very important parts to measure RPU of promoters in iGEM. However, we failed to transfect it to E.coli from parts kit of iGEM. So we decided to amplify this parts by PCR.
 
-
 
-
{|class="experiments"
 
-
!||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10&micro;M)||Primer VR(10&micro;M)||Template E240||KOD Pllus ver.2||Total
 
-
|-
 
-
|E240①||28||3||5||5||1.5||1.5||5||1||50
 
-
|-
 
-
|E240②||28||3||5||5||1.5||1.5||5||1||50
 
-
|}
 
-
 
-
PCR program
 
-
{|class="experiments"
 
-
|94&#x2103;||2min||
 
-
|-
 
-
|98&#x2103;||10sec||rowspan="3"|35 cycles
 
-
|-
 
-
|55&#x2103;||30sec
 
-
|-
 
-
|68&#x2103;||4min
 
-
|-
 
-
|4&#x2103;||forever||
 
-
|}
 
-
 
-
====Electrophoresis====
 
-
 
-
====PCR purification====
 
-
{|class="experiments"
 
-
!Sample number||Concentration(ng/&micro;L)
 
-
|-
 
-
|E240①||42.6
 
-
|-
 
-
|E240②||55.3
 
-
|}
 
-
 
-
====RE====
 
-
To insert E240 to pSB4K5 by 3A assembly, we digested the PCR products of E240 by XbaI and PstI
 
-
{|class="experiments"
 
-
!||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
 
-
|-
 
-
|E240①X-P||30||5||0.5||XbaI||0.2||PstI||0.2||14.1||50
 
-
|-
 
-
|E240②X-P||30||5||0.5||XbaI||0.2||PstI||0.2||14.1||50
 
-
|}
 
-
 
-
====PCR purification====
 
-
{|class="experiments"
 
-
!Sample number||Concentration(ng/&micro;L)||Volume(&micro;L)
 
-
|-
 
-
|E240①X-P||21.8||40
 
-
|-
 
-
|E240②X-P||32.4||45
 
-
|}
 
-
-20&#x2103; freezer
 
-
 
-
====3. Error PCR====
 
-
{|class="experiments"
 
-
!||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
 
-
|-
 
-
|&Delta;TMD①||32||3||5||5||1.5||1.5||1||-||-||1||50
 
-
|-
 
-
|&Delta;TMD②||32||3||5||5||1.5||1.5||-||1||-||1||50
 
-
|-
 
-
|&Delta;TMD③||32||3||5||5||1.5||1.5||-||-||1||1||50
 
-
|}
 
-
 
-
{|class="experiments"
 
-
|94&#x2103;||2min||
 
-
|-
 
-
|98&#x2103;||10sec||rowspan="2"|20 cycles
 
-
|-
 
-
|68&#x2103;||4min
 
-
|-
 
-
|4&#x2103;||forever||
 
-
|}
 
-
 
-
After the digestion by DpnI, we transfected 2&micro;L of sample to 20&micro;L of competent cell.
 
-
 
-
===Tuesday, August 3===
 
-
 
-
====1. The result of transformation====
 
-
{|class="experiments"
 
-
|&Delta;TMD①||Many colonies
 
-
|-
 
-
|&Delta;TMD②||No colony
 
-
|-
 
-
|&Delta;TMD③||Many colonies
 
-
|}
 
-
 
-
We picked two colonies from &Delta;TMD① and &Delta;TMD③, and cultured 37&#x2103; 8/3~8/4.
 
-
 
-
====2. The construction of ML and MS====
 
-
 
-
=====Miniprep=====
 
-
{|class="experiments"
 
-
!Sample number||Concentration(ng/&micro;L)
 
-
|-
 
-
|pSB4K5||60.7
 
-
|-
 
-
|LacP||26.8
 
-
|}
 
-
 
-
=====RE=====
 
-
{|class="experiments"
 
-
!||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
 
-
|-
 
-
|LacP||50||6||0.6||EcoRI||0.2||SpeI||0.2||3||60
 
-
|-
 
-
|pSB4K5(E-P)||50||6||0.6||EcoRI||0.2||PstI||0.2||3||60
 
-
|-
 
-
|E240①(X-P)||50||6||0.6||XbaI||0.2||PstI||0.2||3||60
 
-
|-
 
-
|E240②(X-P)||50||6||0.6||XbaI||0.2||PstI||0.2||3||60
 
-
|}
 
-
Incubate
 
-
 
-
Purification
 
-
 
-
=====PCR purification=====
 
-
{|class="experiments"
 
-
!Sample number||Concentration(ng/&micro;L)
 
-
|-
 
-
|pSB4K5 E-P||39.5
 
-
|-
 
-
|E240①X-P||21.8
 
-
|-
 
-
|E240②X-P||32.4
 
-
|}
 
-
 
-
pSB4K5 E-P is concentrated 10&micro;L and E240①X-P, E240②X-P are concentrated 1&micro;L.
 
-
 
-
Ethanol precipitation
 
-
 
-
1-5-G dilute milliQ 2&micro;L
 
-
 
-
=====Ligation=====
 
-
{|class="experiments"
 
-
!||Vector||Insert 1||Insert 2||Ligation High||Total
 
-
|-
 
-
|ML1||pSB4K5 E-P 1||LacI E-S 1||E240①X-P 1||3||15
 
-
|-
 
-
|ML2||pSB4K5 E-P 1||LacI E-S 1||E240②X-P 1||3||15
 
-
|}
 
-
Incubation 17:30~20:20
 
-
 
-
=====PCR of J23101-E240=====
 
-
 
-
J23101-E240 is important in the measurement of RPU, so we amplified this parts by PCR.
 
-
 
-
{|class="experiments"
 
-
!||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||Primer VF2(10&micro;M)||Primer VR(10&micro;M)||Template J23101-E240||KOD plus ver.2 ||Total
 
-
|-
 
-
|MS①||32||3||5||5||1.5||1.5||1||1||50
 
-
|-
 
-
|MS②||32||3||5||5||1.5||1.5||-||1||50
 
-
|}
 
-
 
-
{|class="experiments"
 
|94&#x2103;||2min||
|94&#x2103;||2min||
|-
|-
Line 558: Line 96:
|4&#x2103;||forever||
|4&#x2103;||forever||
|}
|}
-
 
+
</div>
-
=====PCR purification=====
+
-
{|class="experiments"
+
-
!Sample number||Concentration(ng/&micro;L)
+
-
|-
+
-
|J23101-E240||40.6
+
-
|}
+
-
 
+
-
Discussion
+
-
J23101-E240(MS) is amplified corrrecly.
+
-
 
+
-
=====RE=====
+
-
{|class="experiments"
+
-
!||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
+
-
|-
+
-
|J23101-E240(E-P)||45||6||0.6||EcoRI||0.2||PstI||0.2||8||60
+
-
|}
+
-
 
+
-
=====PCR purification=====
+
-
{|class="experiments"
+
-
!Sample number||Concentration(ng/&micro;L)||Volume(&micro;L)
+
-
|-
+
-
|J23101-E240 E-P ||74.1||30
+
-
|}
+
-
J23101-E240 is concentrated 7&micro;L
+
-
 
+
-
=====Ligation=====
+
-
{|class="experiments"
+
-
!||Vector||Insert||Ligation High||Total
+
-
|-
+
-
|MS||pSB4K5 E-P 1||J23101-E240 E-P 1||2||4
+
-
|}
+
-
Incubation 20:00~20:30
+
-
 
+
-
=====Transformation=====
+
-
{|class="experiments"
+
-
!Sample||Conc(/&micro;L)||Sample Volume(&micro;L)||Competent Cell(&micro;L)||Total||Plate||Incubation
+
-
|-
+
-
|ML1||-||1||20||21||rowspan="3"|LB kan||rowspan="3"|8/3~8/4
+
-
|-
+
-
|ML2||-||1||20||21
+
-
|-
+
-
|MS||-||1||20||21
+
-
|}
+
-
 
+
-
===Thursday, August 5===
+
-
 
+
-
====1. Result of transformation====
+
-
{|class="experiments"
+
-
|ML1||rowspan="3"|Many colonies
+
-
|-
+
-
|ML2
+
-
|-
+
-
|MS
+
-
|}
+
-
 
+
-
ML1 and ML2
+
-
 
+
-
pSB4K5 is inserted RFP generator. We didn't distinguish this inserted parts from low copy plasmid backbone, so self-ligated colony is red.
+
-
So, white colony is correctly inserted parts.
+
-
However, white colonies and green colonies are observed in ML1 and ML2 plate.
+
-
We cultured both white and green colonies.
+
-
 
+
-
MS
+
-
Self-ligated colony is red Many of colonies are red, however,  green colonies are observed.
+
-
 
+
-
=====Culture and Master=====
+
-
 
+
-
Green colony ML1-1 ML1-2 ML2-1 MS1-1 MS1-2 MS1-3
+
-
White colony ML1-3 ML1-4 ML2-2 ML2-3 ML2-4
+
-
 
+
-
J23100 and LacP
+
-
8/5~8/6
+
-
 
+
-
====2. Sequence====
+
-
{|class="experiments"
+
-
!Sample number||Concentration(ng/&micro;L)
+
-
|-
+
-
|&Delta;TMD①A||28.9
+
-
|-
+
-
|&Delta;TMD①B||25.3
+
-
|-
+
-
|&Delta;TMD③A||26.6
+
-
|-
+
-
|&Delta;TMD③B||24.0
+
-
|}
+
-
 
+
-
As a result, deletion is succeeded, however, point mutation is failed. It is because DpnI is too little to digest all of template DNA.
+
-
 
+
-
===Friday, August 6===
+
-
 
+
-
====1. Miniprep====
+
-
ML1-1 ML1-2 ML1-3 ML1-4 ML2-1 ML2-2 ML2-3 ML2-4 MS1 MS2 MS3
+
-
 
+
-
====2. RE====
+
-
{|class="experiments"
+
-
!Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
+
-
|-
+
-
|50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
+
-
|}
+
-
 
+
-
=====Electrophoresis=====
+
-
1 100bp 2&lambda; 3&lambda; 4100bp 5MS1 6MS2 7MS3 8ML1-1 9ML1-2 10ML1-3 11ML1-4 12ML2-1 13ML2-2 14ML2-3 15ML2-4 16MS1 17MS2
+
-
 
+
-
1  2  3  4  5  6  7  8  9  10 11  12 13  14 15 16 17
+
-
 
+
-
[[image:KyotoExp100806-1.png]]
+
-
 
+
-
Discussion
+
-
MS1 and MS2 are inserted correctly. ML1-1 and ML1-2 are inserted correctly. ML2-1 are inserted correctly.
+
-
White colonies are inserted not lacP but its vector. Top10 we used are deleted Lac operon.
+
-
Then, correctly inserted parts is green because of the lack of LacI.
+
-
 
+
-
=====Error PCR(Retry)=====
+
-
{|class="experiments"
+
-
!||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
+
-
|-
+
-
|&Delta;TMD①||32||3||5||5||1.5||1.5||1||1||50
+
-
|-
+
-
|&Delta;TMD②||32||3||5||5||1.5||1.5||1||1||50
+
-
|}
+
-
 
+
-
{|class="experiments"
+
-
|94&#x2103;||2min
+
-
|-
+
-
|98&#x2103;||10sec||rowspan="2"|25 cycles
+
-
|-
+
-
|68&#x2103;||4min
+
-
|-
+
-
|4&#x2103;||forever||
+
-
|}
+
-
 
+
-
Add DpnI 2&micro;L and incubate 1h.
+
-
 
+
-
=====Transformation=====
+
-
{|class="experiments"
+
-
!Sample||Conc(/&micro;L)||Sample Volum(&micro;L)||Competent Cell(&micro;L)||Total||Plate||Incubation
+
-
|-
+
-
|&Delta;TMD①||-||4||50||54||rowspan="3"|LB kan||rowspan="4"|8/6~8/9
+
-
|-
+
-
|&Delta;TMD②||-||4||50||54
+
-
|-
+
-
|2-17-F||-||2||50||52
+
-
|-
+
-
|2-I-5||||2||50||52||LB amp
+
-
|}
+
-
 
+
-
===Monday, August 9===
+
-
By:Wataru, Tomonori, Ken, Takuya
+
-
 
+
-
====1. Miniprep of MS and ML====
+
-
{|class="experiments"
+
-
!Sample number||concentration(ng/&micro;L)
+
-
|-
+
-
|MS||116.2
+
-
|-
+
-
|ML||146.6
+
-
|}
+
-
 
+
-
====2. Transfotrmation of MS and ML====
+
-
{|class="experiments"
+
-
!Sample||conc(ng/&micro;L)||Sample vol(&micro;L)||Competent Cell||Competent cell vol(&micro;L)||Total vol(&micro;L)||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
+
-
|}
+
-
 
+
-
====3. 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&#x2103; 8/9 16:20‾18:20
+
-
 
+
-
After restriction enzyme digestion, we did ethanol precipitation.
+
-
 
+
-
====4. Ligation and Transformation====
+
-
{|class="experiments"
+
-
!Sample||Conc (nu/&micro;L)||Sample vol (&micro;L)||Competent cell||Competent cell vol (&micro;L)||Total vol (&micro;L)||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===
+
-
By: Wataru, Tomonori, Ken, Fumitaka
+
-
 
+
-
====Making culture and Master plate====
+
-
 
+
-
====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 &Delta;TMD1+GFP
+
-
{|class="experiments"
+
-
!Sample number||Concentration (ng/&micro;L)
+
-
|-
+
-
|1-1||9.9
+
-
|-
+
-
|1-2||27.3
+
-
|-
+
-
|2-1||43.2
+
-
|-
+
-
|2-2||34.7
+
-
|}
+
-
37&#x2103; 8/9 18:00‾8/10 9:00
+
-
 
+
-
Culture and Master plate
+
-
 
+
-
===Wednesday, August 11===
+
-
By: Wataru, Naoi, Ken, Takuya
+
-
{|class="experiments"
+
-
!Sample||medium||Cloud||Incubation
+
-
|-
+
-
|rowspan="2"|1||Kanamycin||o||rowspan="14"|37&#x2103;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/&micro;L)
+
-
lac(low)2 : 29.9 (ng/&micro;L)
+
-
 
+
-
====RE and electrophoresis of lac (low) 1 and 3====
+
-
{|class="experiments"
+
-
|Sample name||1||2||3||N
+
-
|-
+
-
|EcoRI||0.2||-||0.2||-
+
-
|-
+
-
|PstI||-||0.2||0.2||-
+
-
|}
+
-
 
+
-
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===
+
-
By: Wataru, Ken
+
-
 
+
-
====RE and electrophoresis of DT====
+
-
{|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===
+
-
By: Wataru, Tomo, Ken
+
-
 
+
-
====1. Miniprep of &Delta;TMD1GFP====
+
-
 
+
-
29.6(ng/&micro;g)
+
-
 
+
-
====2. Point mutation PCR of &Delta;TMD1GFP====
+
-
{|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
+
-
|}
+
-
 
+
-
====3. PCR condition====
+
-
{|class="experiments"
+
-
|94(&#x2103;)||2min||
+
-
|-
+
-
|98||10sec||rowspan="3"|30cycles
+
-
|-
+
-
|55||30sec
+
-
|-
+
-
|68||3.5min
+
-
|-
+
-
|4.0||hold||
+
-
|}
+
-
 
+
-
====. RE(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 r&Delta;TMD1GFP.
+
-
 
+
-
===Monday, August 23===
+
-
By: Wataru, Tomo, Ken, Humitaka, Tasuku
+
-
 
+
-
====1. Miniprep of &Delta;TMD1====
+
-
{|class="experiments"
+
-
|Sample number||Concentration(ng/&micro;g)
+
-
|-
+
-
|1-1||58.9
+
-
|-
+
-
|2-2||49.9
+
-
|}
+
-
 
+
-
====2. Sequencing of &Delta;TMD1 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.
+
-
 
+
-
====3. Screening PCR of SRRz-DT====
+
-
 
+
-
Sample: 1‾13, Marker: lambda and 100, Control:P(1-23L) and N
+
-
 
+
-
PCR condition
+
-
 
+
-
{|class="experiments"
+
-
|90&#x2103;||10min||
+
-
|-
+
-
|94&#x2103;||30sec||rowspan="3"|35cycles
+
-
|-
+
-
|50&#x2103;||30sec
+
-
|-
+
-
|72&#x2103;||1.5min
+
-
|-
+
-
|72&#x2103;||4min||
+
-
|-
+
-
|4&#x2103;||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.
+
-
 
+
-
====4. deletion PCR of r&Delta;TMD1GFP 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
+
-
|}
+
-
 
+
-
PCR condition
+
-
{|class="experiments"
+
-
|94&#x2103;||2min||
+
-
|-
+
-
|94&#x2103;||10sec||rowspan="3"|35cycles
+
-
|-
+
-
|56&#x2103;||30sec
+
-
|-
+
-
|68&#x2103;||3.5min
+
-
|-
+
-
|4&#x2103;||hold||
+
-
|}
+
-
 
+
-
RE (DpnI) and Ligation
+
-
{|class="experiments"
+
-
|Template||25(&micro;L)
+
-
|-
+
-
|DpnI||1
+
-
|-
+
-
|Total||26
+
-
|}
+
-
19:10‾20:10
+
-
 
+
-
{|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 rr&delta;TMD1GFP1, 2 and control.
+
-
 
+
-
===Tuesday, August 24===
+
-
By:Ken, Tomo, Tasuku, Takuya
+
-
 
+
-
====1.Retry of deletion PCR of r&delta;TMD1 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
+
-
|}
+
-
 
+
-
PCR condition
+
-
{|class="experiments"
+
-
|94&#x2103;||2min||
+
-
|-
+
-
|94&#x2103;||10sec||rowspan="3"|35cycles
+
-
|-
+
-
|58&#x2103;||30sec
+
-
|-
+
-
|68&#x2103;||3.5min
+
-
|-
+
-
|4&#x2103;||hold||
+
-
|}
+
-
 
+
-
RE (DpnI), electrophoresis and ligation
+
-
RE: 14:15‾15:15
+
-
Electrophoresis:
+
-
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.
+
-
 
+
-
====2.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
+
-
|-
+
-
|}
+
-
 
+
-
PCR condition
+
-
{|class="experiments"
+
-
|94&#x2103;||2min||
+
-
|-
+
-
|98&#x2103;||10sec||rowspan="3"|30cycles
+
-
|-
+
-
|55&#x2103;||30sec
+
-
|-
+
-
|68&#x2103;||4min
+
-
|-
+
-
|4&#x2103;||hold||
+
-
|}
+
-
 
+
-
RE(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
+
-
|-
+
-
|}
+
-
 
+
-
Purification:
+
-
　   Sample1: 5.5*50(ng/&micro;L)
+
-
　   Sample2: 5.2*50(ng/&micro;L)
+
-
 
+
-
RE(EcoRI, PstI) and Gel extraction
+
-
　   Sample1: 28.8 (ng/&micro;L)
+
-
　   Sample2: 26.4 (ng/&micro;L)
+
-
 
+
-
Transformation
+
-
Sample: rr&Delta;TMD1GFP1. 2. control, and rSRRz1. 2. control
+
-
 
+
-
 
+
-
===Wednesday, August 25===
+
-
By:Ken, Tomo, Kazuya, Tasuku, Takuya
+
-
 
+
-
Making culture and Master plate
+
-
{|class="experiments"
+
-
|rr&Delta;TMD1-1||rowspan="2"|Many Colonies
+
-
|-
+
-
|rr&Delta;TMD1-2
+
-
|-
+
-
|rr&Delta;TMD1-C-||zero
+
-
|-
+
-
|rSRRz-1||rowspan="2"|Many Colonies
+
-
|-
+
-
|rSRRz-2
+
-
|-
+
-
|rSRRz-C-||zero
+
-
|}
+
-
 
+
-
Miniprep of 1-5G
+
-
29.0 (ng/&micro;L)
+
-
 
+
-
RE 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/&micro;L)
+
-
|-
+
-
|1-5G||18.4
+
-
|-
+
-
|Lac low||8.6
+
-
|}
+
-
 
+
-
Ligation and transformation
+
-
Ligation of E0240 and 1-5G
+
-
 
+
-
 
+
-
===Thursday, August 26===
+
-
By:Ken, Tomo, Kazuya, Tasuku, Takuya, Hashiya
+
-
 
+
-
Miniprep
+
-
{|class="experiments"
+
-
|Sample name||Concentration(ng/&micro;L)
+
-
|-
+
-
|constP(0.7)||44.5
+
-
|}
+
-
 
+
-
RE 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/&micro;L
+
-
 
+
-
===Friday, August 27===
+
-
By:Ken, Tomo, Kazuya, Hashiya
+
-
 
+
-
Making master plate of E0240 low
+
-
 
+
-
{|class="experiments"
+
-
|Sample Name||Concentration(ng/&micro;L)
+
-
|-
+
-
|rr&Delta;TMD1 1-2||20.9
+
-
|-
+
-
|rSRRz 1-1||16.4
+
-
|}
+
-
 
+
-
RE of rr&Delta;TMD1 and rSRRz
+
-
{|class="experiments"
+
-
!Sample name||Template||10xbuffer||100xbuffer||XbaI||PstI||Water||Total
+
-
|-
+
-
|rr&Delta;TMD1 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/&micro;L)
+
-
|-
+
-
|rr&Delta;TMD1 1-2||44.7
+
-
|-
+
-
|rSRRz 1-1||56.1
+
-
|}
+
-
 
+
-
Lagation and transformation
+
-
lacP + rr&Delta;TMD1 1-2
+
-
constP (0.7) + rr&Delta;TMD1 1-2
+
-
lac low + rSRRz 1-1
+
-
 
+
-
 
+
-
===Monday, August 30===
+
-
By: Tomonori, Kazuya, Tasuku, Ken
+
-
====Making culture and Master plate====
+
-
{|class="experiments"
+
-
|lacP rr&Delta;TMD1GFP||Many colonies
+
-
|-
+
-
|lacP rr&Delta;TMD1GFP(control)||Some colonies
+
-
|-
+
-
|constP rr&Delta;TMD1GFP||Many colonies
+
-
|-
+
-
|constP rr&Delta;TMD1GFP(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===
+
-
By: Tomonori Y, Takuya, Kazuya, Tasuku,Takuya, Ken
+
-
====Miniprep====
+
-
{|class="experiments"
+
-
|constP (0.3)||48.5 (ng/&micro;L)
+
-
|-
+
-
|lac rr&Delta;TMD1||107.3
+
-
|}
+
-
 
+
-
====RE of constP (0.3) and lac rr&Delta;TMD1====
+
-
 
+
-
====Gel Extraction of lac rr&Delta;TMD1====
+
-
 
+
-
[[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 rr&Delta;TMD1====
+
-
{|class="experiments"
+
-
|constP (0.3)||5.8 (ng/&micro;L)
+
-
|-
+
-
|lac rr&Delta;TMD1||7.8 (ng/&micro;L)
+
-
|}
+
-
 
+
-
====Ligation and transformation====
+
-
{|class="experiments"
+
-
|Insert||Vector
+
-
|-
+
-
|lac rr&Delta;TMD1||constP (0.3)
+
-
|}
+
-
 
+
-
===Wednesday, September 1===
+
-
By: Tomonori, Kazuya, Tasuku, Humitaka, Ken
+
-
Making culture and Master plate
+
-
{|class="experiments"
+
-
|lac rr&Delta;TMD1 constP||many colonies
+
-
|-
+
-
|lac rr&Delta;TMD1 const (control)||many colonies
+
-
|}
+
-
 
+
-
Screenig PCR of lacP-rr&Delta;TMD1GFP-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/&micro;L)
+
-
|-
+
-
|low||56.0 (ng/&micro;L)
+
-
|}
+
-
 
+
-
====RE 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/&micro;L)
+
-
|-
+
-
|low||16.8
+
-
|}
+
-
 
+
-
====Ligation and transformation====
+
-
Insert: rSRRz 1-1 Vector: low copy plasmid
+
-
 
+
-
 
+
-
===9/2===
+
-
By: Tomonori, Tomo, Takuya, Ken
+
-
 
+
-
====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.
+
-
 
+
-
 
+
-
 
+
-
===9/3===
+
-
By: Tomonori, Tomo, Kazuya, Tasuku, Humitaka, Ken
+
-
 
+
-
====Making culture====
+
-
lac rr&Delta;TMD1 1, 3
+
-
rr&Delta;TMD1 1-1, 1-2
+
-
rSRRz 1-1, 1-2
+
-
ML
+

Revision as of 12:13, 30 September 2010

Contents

Index

Notebook

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

Solubilization of Antibiotics

Ampicillin(Amp)Kanamycin(Kan)
Mix 1.0g Amp and 20ml MilliQMix 0.5g Kan and 10ml MilliQFinal concentration is 50mg/ml
Dispense 1.1ml of the solution into 1.5ml tubes
Store in the freezer (-20℃)

Making plates for LB (Amp+) and LB (Kan+)

Transformation

NameWellSample (µl)Competent Cells (µl)Total (µl)PlateIncubationResult
<partinfo>J23100</partinfo>1-18-C12021LB (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 "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Amp+). And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance. So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".

Wednesday, July 21 By: Wataru, Ken, Makoto, Takuya Y.

Culture of plates in which colonies was observed at 37℃ from 07/21 20:50 to 07/22 17:00

Making a master plate of the above plates

=Retry Transformation

!Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result |- |<partinfo>pSB4K5</partinfo>||1-5-G||1||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||○ |}

PCR for S-R-Rz/Rz1 and S

No.Water25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2TemplateDNA (5ng/µl)Primer Forward (10µM)Primer S-R-Rz/Rz1 Reverse (10µM)Primer S Reverse (10µM)KOD Plus ver.2Total
128µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
228µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
328µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
428µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
528µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
628µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
728µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
828µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
PCR condition
94℃2min
98℃10sec30 cycles
55℃30sec
68℃4min
4℃forever
Retrieved from "http://2010.igem.org/Team:Kyoto/Notebook"