Team:Kyoto/Notebook

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{{:Team:Kyoto/Header}}
{{:Team:Kyoto/Header}}
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==Index==
 
==Notebook==
==Notebook==
 +
===Notebooks===
 +
* [[Team:Kyoto/Notebook1|Notebook1]]: Construction for Lysisbox.
 +
* [[Team:Kyoto/Notebook2|Notebook2]]: Measurement of R0011.
 +
* [[Team:Kyoto/Notebook3|Notebook3]]: Measurement of Lysis Cassette and Lysisbox etc.
-
<div class="note">
+
[[#top-section|^Top]]
-
===Tuesday, July 20===
+
===Other Information===
 +
* [[Team:Kyoto/Protocols|Protocols]]: Protocols of each experiment such as Polymerase Chain Reaction (PCR), Restriction Digestion, Ligation, Transformation.
 +
* [[Team:Kyoto/Materials|Materials]]: Strains, DNA, and Primers.
 +
* [[Team:Kyoto/Parts|Parts]]: Construction of each part and BioBrick Parts used in our project.
-
By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
+
[[#top-section|^Top]]
-
====1. Solubilization of antibiotics.====
 
-
 
-
For Ampicillin(Amp): add 1.0g Amp to 20ml MilliQ (final concentration is 50mg/ml).
 
-
 
-
For Kanamycin(kan): add 0.5g Kan to 10ml MilliQ (final concentration is 50mg/ml).
 
-
 
-
Dispense 1.1ml of the solution into 1.5ml tubes.
 
-
 
-
Store in the freezer (-20&#x2103;).
 
-
 
-
====2. Making plates for LB (Amp+) and LB (Kan+).====
 
-
 
-
====3. [[Team:Kyoto/Protocols#Transformation|Transformation]] of iGEM Parts.====
 
-
 
-
{| class="experiments"
 
-
!Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
 
-
|-
 
-
|<partinfo>J23100</partinfo>||1-18-C||1||20||21||rowspan="7"|LB (Amp+)||rowspan="8"|At 37&#x2103; 7/20 20:50 - 7/21 17:00||○
 
-
|-
 
-
|<partinfo>J23105</partinfo>||1-18-M||1||20||21||○
 
-
|-
 
-
|<partinfo>J23116</partinfo>||1-20-M||1||20||21||○
 
-
|-
 
-
|<partinfo>R0011</partinfo>||1-6-G||1||20||21||○
 
-
|-
 
-
|<partinfo>E0840</partinfo>||1-12-O||1||20||21||○
 
-
|-
 
-
|<partinfo>J06702</partinfo>||2-8-E||1||20||21||○
 
-
|-
 
-
|<partinfo>pSB4K5</partinfo>||1-5-G||1||20||21||×
 
-
|-
 
-
|<partinfo>B0015</partinfo>||1-23-L||1||20||21||LB (Kanamycin+)||×
 
-
|}
 
-
 
-
A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Ampicillin+). And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance. So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".
 
-
 
-
</div>
 
-
 
-
<div class="note">
 
-
===Wednesday, July 21===
 
-
 
-
By: Wataru, Ken, Makoto, Takuya Yamamoto
 
-
 
-
====1. Culture plates in which colonies was observed at 37&#x2103; from 07/21 20:50 to 07/22 17:00.====
 
-
 
-
====2. Make a master plate of the above plates.====
 
-
 
-
====3. Retry [[Team:Kyoto/Protocols#Transformation|Transformation]] of iGEM Parts.====
 
-
 
-
{| class="experiments"
 
-
!Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
 
-
|-
 
-
|<partinfo>pSB4K5</partinfo>||1-5-G||1||20||21||rowspan="2"|LB (Kanamycin+)||rowspan="2"|At 37℃ 7/21 20:50 - 7/22 16:30||○
 
-
|-
 
-
|<partinfo>B0015</partinfo>||1-23-L||1||20||21||○
 
-
|}
 
-
 
-
====4. [[Team:Kyoto/Protocols#PCR|PCR]] for S-R-Rz/Rz1 and S====
 
-
 
-
Dilute &lambda;DNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template &lambda;DNA was 5ng/µl.
 
-
 
-
{| class="experiments"
 
-
!No.||Water||25mM MgSO4||2mM dNTPs||10xBuffer for KOD Plus ver.2||TemplateDNA (5ng/µl)||Primer S-R-Rz/Rz1 Forward (10µM)||Primer S-R-Rz/Rz1 Reverse (10µM)||Primer S Reverse (10µM)||KOD Plus ver.2||Total
 
-
|-
 
-
|1||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
-
|-
 
-
|2||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
-
|-
 
-
|3||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 
-
|-
 
-
|4||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 
-
|-
 
-
|5||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
-
|-
 
-
|6||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
-
|-
 
-
|7||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|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|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-GFP<sub>1</sub>||<partinfo>E0840</partinfo> 0.5&micro;l||S<sub>1</sub> 0.5||1||2
 
-
|-
 
-
|S-GFP<sub>2</sub>||<partinfo>E0840</partinfo> 0.5||S<sub>2</sub> 0.5||1||2
 
-
|}
 
-
 
-
</div>
 
-
 
-
<div class="note">
 
-
 
-
===Monday, July 26===
 
-
 
-
By: Wataru, Tomonori, Makoto
 
-
 
-
====1. Electrophoresis of PCR products====
 
-
 
-
[[Image:KyotoExp100726-1.png|right]]
 
-
 
-
At the condition 4 (4.5&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||
 
-
|-
 
-
|5||59.3||
 
-
|-
 
-
|6||59.6||
 
-
|}
 
-
 
-
====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>
 
----
----
-
 
-
===Monday, July 26===
 
-
By: Wataru, Tomonari, Makoto
 
-
 
-
====Electrophoresis of PCR products====
 
-
 
-
3①3②4.5①4.5②6①6②
 
-
 
-
[[image : KyotoExp100726-1.png]]
 
-
 
-
Discussion
 
-
 
-
At the condition 4.5②　and 6②、S-R-Rz is amplified very much. So we decided to use them inexperience.
 
-
 
-
====PCR purification====
 
-
{|class="wikitable"
 
-
!Sample number||Concentration(ng/&micro;L)
 
-
|-
 
-
|4.5②||51.6
 
-
|-
 
-
|6①||59.3
 
-
|-
 
-
|6②||59.6
 
-
|}
 
-
 
-
====Transformation====
 
-
{|class="wikitable"
 
-
!Sample||Conc(/&micro;L)||Sample Volume(&micro;L)||Competent Cell(&micro;L)||Total||Plate||Incubation
 
-
|-
 
-
|1-12-M||-||1||20||21||LB amp||rowspan="3"|7/26~7/27
 
-
|-
 
-
|2-17-F||-||1||20||21||rowspan="2"|LB kan
 
-
|-
 
-
|1-5-E||-||1||20||21
 
-
|}
 
-
 
-
====Culture====
 
-
 
-
1-6-G, 1-12-O, 1-23-L
 
-
 
-
===Tuesday, July 27===
 
-
By: Wataru, Tomo, Kazuya, Ken, Naoi
 
-
 
-
Result of transformation
 
-
{|class="wikitable"
 
-
|1-12-M||rowspan="3"|No colony
 
-
|-
 
-
|1-5-E
 
-
|-
 
-
|2-17-F
 
-
|}
 
-
 
-
====Colony of PCR of S-E840====
 
-
 
-
To check that S GFP is correctly inserted, we did colony PCR.
 
-
 
-
Gel: Agarose
 
-
Time: 35min
 
-
Voltage: 100V
 
-
Maker: 1K 100
 
-
1～6 S-GFP①
 
-
7～13 S-GFP②
 
-
Posi  E840（about 900bp）
 
-
Nega None
 
-
 
-
1K  1  2  3  4  5  6  7  8  9  10  11  12  13 posi nega 100
 
-
[[image:KyotoExp100727.png]]
 
-
 
-
As a result, 1,3,5,6,11,12,13 are inserted S gene correctly.
 
-
So, we decided to use 6 as S-E840①　and 11 as S-E840②.
 
-
 
-
====Miniprep====
 
-
{|class="wikitable"
 
-
!Sample number||Concentration(ng/&micro;L)
 
-
|-
 
-
|1-6-G||26.9
 
-
|-
 
-
|1-23-L||120.0
 
-
|-
 
-
|1-12-O||120.1
 
-
|}
 
-
 
-
====RE====
 
-
{|class="wikitable"
 
-
!||Sample volume||2 buffer||BSA||colspan="2"|Enzyme 1||colspan="2"|Enzyme 2||MilliQ||Total
 
-
|-
 
-
|1-23-L||30||5||0.5||rowspan="3"|EcoRI||0.4||rowspan="3"|XbaI||0.3||13.7||50
 
-
|-
 
-
|4.5②||40||5||0.5||0.4||0.4||3.8||50
 
-
|-
 
-
|6②||40||5||0.5||0.4||0.4||3.8||50
 
-
|}
 
-
Incubate 37&#x2103; 16:45~18:00
 
-
 
-
====Ligation====
 
-
 
-
====Transformation====
 
-
 
-
===Wednesday, July 28===
 
-
 
-
====1. Result of Transformation====
 
-
{|class="wikitable"
 
-
|SRRz①-DT||rowspan="2"|Many colonies
 
-
|-
 
-
|SRRz②-DT
 
-
|}
 
-
 
-
====2. Deletion PCR====
 
-
To delete functional domain of S gene, we did deletion PCR.
 
-
 
-
=====Miniprep=====
 
-
{|class="wikitable"
 
-
!Sample number||Concentration(ng/&micro;)
 
-
|-
 
-
|S-E840①||95.5
 
-
|-
 
-
|S-E840②||98.6
 
-
|}
 
-
 
-
Diluted S-GFP①　and S-GFP②　20 times with water, and used as template DNA.
 
-
 
-
=====Deletion PCR=====
 
-
 
-
To delete functional region of S gene, we did deletion PCR.
 
-
 
-
{|class="wikitable"
 
-
!||Water||25mM MgSO4||2mM dNTPs||10x buffer for KOD Plus ver.2||Primer Deletion F(10&micro;M)||Primer Deletion R(10&micro;M)||Template S-E840①||Template S-E840②||KOD Plus ver.2||Total
 
-
|-
 
-
|&Delta;1-1||28||3||5||5||1.5||1.5||5||-||1||50
 
-
|-
 
-
|&Delta;1-2||28||3||5||5||1.5||1.5||5||-||1||50
 
-
|-
 
-
|&Delta;2-1||28||3||5||5||1.5||1.5||-||5||1||50
 
-
|}
 
-
 
-
PCR program
 
-
{|class="wikitable"
 
-
|94&#x2103;||2min||
 
-
|-
 
-
|98&#x2103;||10sec||rowspan="3"|35 cycles
 
-
|-
 
-
|55&#x2103;||30sec
 
-
|-
 
-
|-
 
-
|68&#x2103;||4min
 
-
|-
 
-
|4&#x2103;||forever||
 
-
|}
 
-
 
-
====3. RE====
 
-
To check function of our Restriction enzymes, we digested S-E840① and S-E-840② by DpnI.
 
-
{|class="wikitable"
 
-
!||Sample||fast digestion buffer||DpnI||MilliQ||Total
 
-
|-
 
-
|S-E840①||3||1||0.1||5.8||10
 
-
|-
 
-
|S-E840②||3||1||0.1||5.8||10
 
-
|}
 
-
 
-
=====Electrophoresis=====
 
-
Gel: Agarose
 
-
Time: 35min
 
-
Voltage: 100V
 
-
Maker: 1K 100
 
-
 
-
1 1k marker 2 not digested S-E840① 3  not digested S-E840② 4 digested S-E840①  5 digested S-E840② 6 100bp marker
 
-
 
-
1k  1  2  3  4  100
 
-
 
-
[[image:KyotoExp100728.png]]
 
-
 
-
Discussion
 
-
 
-
DpnI works correctly
 
-
 
-
===Thursday, July 29===
 
-
 
-
====RE====
 
-
{|class="wikitable"
 
-
!||Sample volume||Fastdigestion buffer||Enzyme 1||MilliQ||Total
 
-
|-
 
-
|&Delta;1-1||50||6||DpnI 0.2||3.8||60
 
-
|-
 
-
|&Delta;2-1||50||6||DpnI 0.2||3.8||60
 
-
|}
 
-
Incubate 7/29 9:40~7/29 11:00
 
-
 
-
====Ligation and Pospholylation====
 
-
{|class="wikitable"
 
-
!||Sample||MilliQ||Ligation High||T4 Kinase||Total
 
-
|-
 
-
|&Delta;1-1||2||7||5||1||15
 
-
|-
 
-
|&Delta;2-1||2||7||5||1||15
 
-
|}
 
-
Incubate 7/29 11:30~7/29 13:00
 
-
 
-
====Transformation====
 
-
{|class="wikitable"
 
-
!Sample||Conc(/&micro;L)||Sample Volume(&micro;L)||Competent Cell(&micro;L)||Total||Plate||Incubation
 
-
|-
 
-
|&Delta;1-1||-||3||30||33||rowspan="2"|LB amp||rowspan="2"|7/29~7/30
 
-
|-
 
-
|&Delta;1-1||-||3||30||33
 
-
|}
 
-
 
-
===Friday, July 30===
 
-
Result of transformation of &Delta;1 and &Delta;2
 
-
Many colonies are observed.
 

Latest revision as of 11:33, 27 October 2010

Contents

Notebook

Notebooks

^Top

Other Information

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

^Top

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