Team:Kyoto/Notebook

From 2010.igem.org

(Difference between revisions)
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{{:Team:Kyoto/Header}}
{{:Team:Kyoto/Header}}
==Index==
==Index==
-
 
==Notebook==
==Notebook==
-
 
<div class="note">
<div class="note">
===Tuesday, July 20===
===Tuesday, July 20===
By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
-
 
====1. Solubilization of antibiotics.====
====1. Solubilization of antibiotics.====
* for Ampicillin(Amp): add 1.0g Amp to 20ml MilliQ (final concentration is 50mg/ml).
* for Ampicillin(Amp): add 1.0g Amp to 20ml MilliQ (final concentration is 50mg/ml).
Line 13: Line 10:
* 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"
Line 41: Line 36:
* 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".
 +
</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]]
 +
* '''Discussion'''
 +
* 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
 +
|}
 +
* '''Discussion'''
 +
* 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>||-
 +
|}
 +
* '''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.
 +
====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>
 +
|}
 +
* '''Discussion'''
 +
* 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]]
 +
* '''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.
 +
====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]]
 +
* '''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.
 +
====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>
----
----

Revision as of 04:41, 8 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".

Wednesday, July 21

By: Wataru, Ken, Makoto, Takuya Yamamoto

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

2. Make a master plate of the above plates.

3. Retry Transformation of iGEM Parts.

NameWellSample (µl)Competent Cells (µl)Total (µl)PlateIncubationResult
<partinfo>pSB4K5</partinfo>1-5-G12021LB (Kanamycin+)At 37℃ 7/21 20:50 - 7/22 16:30○
<partinfo>B0015</partinfo>1-23-L12021○

4. PCR for S-R-Rz/Rz1 and S

  • Dilute λDNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template λDNA was 5ng/µl.
No.Water25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2TemplateDNA (5ng/µl)Primer S-R-Rz/Rz1 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
  • Forward Primer of S-R-Rz/Rz1 and S is common.
  • PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.

Thursday, July 22

By: Wataru

1. Electrophoresis of the PCR products for 40min.

File:KyotoEXP100722-1.png
  • Discussion
  • Length of S and S-R-Rz/Rz1 is 370bp, 1300bp, so PCR succeeded.

2. Miniprep of iGEM Parts.

NameConcentration(ng/µl)
<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
  • Discussion
  • 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.

Friday, July 23

By: Wataru, Tomo, Makoto

1. Miniprep of iGEM Parts.

NameConcentration(ng/µl)
<partinfo>pSB4K5</partinfo>79.2
<partinfo>B0015</partinfo>-
  • 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.

2. Picked up 1, 3, 5, 7 of the products of PCR, and purified by PCR-purification.

SampleConcentration (ng/µl)New Name
118.6-
377.6S1
533.6-
765.4S2
  • Discussion
  • 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.

SampleWater25mmol/l MgSO42mmol/l dNTPs10×Buffer for KOD plus ver.2Template DNA (5ng/µl)Primer S-R-Rz/Rz1 Forward (10µmol/l)Primer S-R-Rz/Rz1 Reverse (10µmol/l)KOD plus ver.2Total
128µl35551.51.5150
22835551.51.5150
326.54.55551.51.5150
426.54.55551.51.5150
52565551.51.5150
62565551.51.5150
  • PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.

4. Digested <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, PstI to check function of our Restriction enzymes.

Sample10xBufferBSAEnzymeMilliQTotalIncubation
15µl1EcoRI 0.13.610At 37℃ 7/23 18:00 - 7/23 18:30
251XbaI 0.13.610
351SpeI 0.13.610
451PstI 0.13.610
551-3.710

|-

5. Electrophoresis of above sample for 35min.

KyotoExp100723-1.png
  • 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.

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.

Sample10×BufferEnzyme 1Enzyme 2MilliQTotalIncubation
S111µl5EcoRI 0.2SpeI 0.233.650At 37℃ for 2h
S2115EcoRI 0.2SpeI 0.233.650
<partinfo>E0840</partinfo>(GFP)455EcoRI 0.2XbaI 0.2050
  • After PCR purification, evaporated them and diluted 3ul.

7. Ligated over night.

SampleVectorInsertLigation HighTotal
S-GFP1<partinfo>E0840</partinfo> 0.5µlS1 0.512
S-GFP2<partinfo>E0840</partinfo> 0.5S2 0.512

Monday, July 26

By: Wataru, Tomonori, Makoto

1. Electrophoresis of PCR products

KyotoExp100726-1.png
  • Discussion
  • At the condition 4 (4.5µl MgSO4) and 6 (6µl MgSO4), S-R-Rz/Rz1 is amplified very much. So we decided to use them.

2. PCR purification

SampleConcentration (ng/µl)New Name
451.6
559.3
659.6

3. Transformation of iGEM Parts

NameWellSample (µl)Competent Cell (µl)Total (µl)PlateIncubationResult
1-12-M12021LB (Amplicillin+)At 37℃ 7/26 - 7/27×
2-17-F12021LB (Kanamycin+)×
1-5-E12021×

4. Culture of 1-6-G, 1-12-O, and 1-23-L=

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