Team:Kyoto/Notebook

From 2010.igem.org

(Difference between revisions)
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==Notebook==
==Notebook==
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=== ===
+
<div class="note">
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{| class="note"
+
===Tuesday, July 20===
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|+ Tuesday, July 20
+
By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
-
|-
+
 
-
| 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).
-
| (1) Solubilization of antibiotics.
+
* for Kanamycin(kan): add 0.5g Kan to 10ml MilliQ (final concentration is 50mg/ml).
-
* 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.
* Dispense 1.1ml of the solution into 1.5ml tubes.
* Store in the freezer (-20&#x2103;).
* Store in the freezer (-20&#x2103;).
-
|-
+
 
-
| (2) Making plates for LB (Amp+) and LB (Kan+).
+
====2. Making plates for LB (Amp+) and LB (Kan+).====
-
|-
+
 
-
| (3) [[Team:Kyoto/Protocols#Transformation|Transformation]] of iGEM Parts.
+
====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
!Name||Well||Sample (µl)||Competent Cells (µl)||Total (µl)||Plate||Incubation||Result
|-
|-
-
|<partinfo>J23100</partinfo>||1-18-C||1||20||21||rowspan="7"|LB (Ampicillin+)||rowspan="8"|At 37&#x2103; 7/20 20:50 - 7/21 17:00||○
+
|<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>J23105</partinfo>||1-18-M||1||20||21||○
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* And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance.
* 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".
* So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".
-
|}
 
----
----
-
 
-
=== ===
 
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{| class="note"
 
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|+Wednesday, July 21
 
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|-
 
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|
 
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*By: Wataru, Ken, Makoto, Takuya Yamamoto
 
-
*Category: Transformation, PCR, Lysis Cassette
 
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|-
 
-
|(1) Cultured plates in which colonies was observed at 37&#x2103; from 07/21 20:50 to 07/22 17:00.
 
-
|-
 
-
|(2) Made a master plate of the above plates.
 
-
|-
 
-
|(3) Retried [[Team:Kyoto/Protocols#Transformation|Transformation]] of iGEM Parts.
 
-
{| class="experiments"
 
-
|Name||Well<sup>*1</sup>||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||○
 
-
|}
 
-
* *1 "1-5-G" means well 5G in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
 
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|-
 
-
|(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
 
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|-
 
-
|1||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
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|-
 
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|2||28µl||3µl||5µl||5µl||5µl||1.5µl||1.5µl||-||1µl||50µl
 
-
|-
 
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|3||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 
-
|-
 
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|4||28µl||3µl||5µl||5µl||5µl||1.5µl||-||1.5µl||1µl||50µl
 
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|-
 
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|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.
 
-
|}
 
-
----
 
-
 
-
=== ===
 
-
{| class="note"
 
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|+ Thursday 22, July
 
-
|-
 
-
|
 
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*By: Wataru
 
-
*Category: Lysis Cassette, parts
 
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|-
 
-
|(1) Electrophoresis of the PCR products for 40min.
 
-
[[Image:KyotoExp100722-1.png|right]]
 
-
* '''Discussion'''
 
-
* Length of S and S-R-Rz/Rz1 is 370bp, 1300bp, so PCR succeeded.
 
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|-
 
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|(2) [[Team:Kyoto/Protocols#Miniprep|Miniprep]] of iGEM Parts.
 
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{| class="experiments"
 
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!Name||Concentration(ng/&micro;l)
 
-
|-
 
-
|<partinfo>J23100</partinfo>||18.5
 
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|-
 
-
|<partinfo>J23105</partinfo>||12.5
 
-
|-
 
-
|<partinfo>J23116</partinfo>||14.6
 
-
|-
 
-
|<partinfo>R0011</partinfo>||8.6
 
-
|-
 
-
|<partinfo>E0840</partinfo>||12.1
 
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|-
 
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|<partinfo>J06702</partinfo>||14.7
 
-
|}
 
-
* '''Discussion'''
 
-
* The concentration of all samples was very week. Probably our shaking incubation was week.
 
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|-
 
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|(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.
 
-
|}
 
-
----
 
-
 
-
=== ===
 
-
{| class="note"
 
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|+ Friday 23, July
 
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|
 
-
* By: Wataru, Tomo, Makoto
 
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* Category:
 
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|-
 
-
|(1) [[Team:Kyoto/Protocols#Miniprep|Miniprep]] of iGEM Parts.
 
-
{| class="experiments"
 
-
!Name||Concentration(ng/&micro;l)
 
-
|-
 
-
|<partinfo>pSB4K5</partinfo>||79.2
 
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|-
 
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|<partinfo>B0015</partinfo>||-
 
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|}
 
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* '''Discussion'''
 
-
* 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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|-
 
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|(2) Picked up 1, 3, 5, 7 of the products of PCR, and purified by PCR-purification.
 
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{| class="experiments"
 
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!Sample||Concentration (ng/&micro;l)||New Name||
 
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|-
 
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|1||18.6||-
 
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|-
 
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|3||77.6||S<sub>1</sub>
 
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|-
 
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|5||33.6||-
 
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|-
 
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|7||65.4||S<sub>2</sub>
 
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|}
 
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* '''Discussion'''
 
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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.
 
-
|-
 
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|(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
 
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|-
 
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|1||28&micro;l||3||5||5||5||1.5||1.5||1||50
 
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|-
 
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|2||28||3||5||5||5||1.5||1.5||1||50
 
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|-
 
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|3||26.5||4.5||5||5||5||1.5||1.5||1||50
 
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|-
 
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|4||26.5||4.5||5||5||5||1.5||1.5||1||50
 
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|-
 
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|5||25||6||5||5||5||1.5||1.5||1||50
 
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|-
 
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|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.
 
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|-
 
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|(4) Digested <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, PstI to check function of our Restriction enzymes.
 
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{| class="experiments"
 
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!Sample||10xBuffer||BSA||Enzyme||MilliQ||Total||Incubation
 
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|-
 
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|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
 
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|-
 
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|2||5||1||''Xba''I 0.1||3.6||10
 
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|-
 
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|3||5||1||''Spe''I 0.1||3.6||10
 
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|-
 
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|4||5||1||''Pst''I 0.1||3.6||10
 
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|-
 
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|5||5||1||-||3.7||10
 
-
|}
 
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|-
 
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|(5) Electrophoresis of above sample for 35min.
 
-
[[Image:KyotoExp100723-1.png|right]]
 
-
* '''Discussion'''
 
-
* 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.
 
-
|-
 
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|(6) To insert S gene to GFP, we digested the PCR products of S gene by EcoRi and SpeI, and GFP by EcoRl and XbaI.
 
-
{| class="experiments"
 
-
!Sample||10×Buffer||Enzyme 1||Enzyme 2||MilliQ||Total||Incubation
 
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|-
 
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|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
 
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|-
 
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|S<sub>2</sub>||11||5||''EcoR''I 0.2||''Spe''I 0.2||33.6||50
 
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|-
 
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|<partinfo>E0840</partinfo>(GFP)||45||5||''EcoR''I 0.2||''Xba''I 0.2||0||50
 
-
|}
 
-
* After PCR purification, evaporated them and diluted 3ul.
 
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|-
 
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|Ligated over night
 
-
{| class="experiments"
 
-
!Sample||Vector||Insert||Ligation High||Total
 
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|-
 
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|S-GFP<sub>1</sub>||<partinfo>E0840</partinfo> 0.5&micro;l||S<sub>1</sub> 0.5||1||2
 
-
|-
 
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|S-GFP<sub>2</sub>||<partinfo>E0840</partinfo> 0.5||S<sub>2</sub> 0.5||1||2
 
-
|}
 
-
|}
 
-
----
 
-
 
-
=== ===
 
-
{| class="note"
 
-
|+ Monday, July 26
 
-
|
 
-
* By: Wataru, Tomonori, Makoto
 
-
* Category:
 
-
|-
 
-
|(1) Electrophoresis of PCR products
 
-
[[Image:KyotoExp100726-1.png|right]]
 
-
* '''Discussion'''
 
-
* 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.
 
-
|-
 
-
|PCR purification
 
-
{| class="experiments"
 
-
!Sample||Concentration (ng/&micro;l)||New Name
 
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|-
 
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|4||51.6||
 
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|-
 
-
|5||59.3||
 
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|-
 
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|6||59.6||
 
-
|}
 
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|-
 
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|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+)||×
 
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|-
 
-
|||1-5-E||1||20||21||×
 
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|}
 
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|-
 
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|Culture of 1-6-G, 1-12-O, and 1-23-L
 
-
|}
 

Revision as of 19:11, 6 September 2010

Contents

Index

Notebook

Tuesday, July 20

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.
  • Store in the freezer (-20℃).

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

3. Transformation of iGEM Parts.

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 (Kanamycin+)×
  • "1-18-C" means well 18C in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
  • Discussion
  • 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".
Retrieved from "http://2010.igem.org/Team:Kyoto/Notebook"