Team:Kyoto/Notebook

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|-
|-
|4℃||forever||
|4℃||forever||
 +
|}
 +
</div>
 +
===Thursday, July 22 <span class="by">By: Wataru</span>===
 +
<div class="electrophoresis lysis">
 +
====[[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 and 1300bp, so PCR succeeded.
 +
</div>
 +
----
 +
<div class="miniprep lysis measure">
 +
====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
 +
{| 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.
 +
</div>
 +
----
 +
<div class="culture lysis">
 +
====Culture of plates and making master plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo> from 07/22 17:00 to 07/23 10:00====
 +
</div>
 +
===Friday, July 23 <span class="by">By: Wataru, Tomo, Makoto</span>===
 +
<div class="miniprep lysis">
 +
====[[Team:Kyoto/Protocols#Miniprep|Miniprep]]====
 +
{| 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.
 +
</div>
 +
----
 +
<div class="pcr-purification lysis">
 +
====Picking up number 1, 3, 5, and 7 of the products of PCR, and PCR-purification====
 +
{| class="experiments"
 +
!No.||Name||Concentration (ng/&micro;l)||New Name
 +
|-
 +
|1||S-R-Rz/Rz1||18.6||-
 +
|-
 +
|3||S||77.6||S<sub>1</sub>
 +
|-
 +
|5||S-R-Rz/Rz1||33.6||-
 +
|-
 +
|7||S||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.
 +
</div>
 +
----
 +
<div class="pcr lysis">
 +
====Retry of [[Team:Kyoto/Protocols#Standard_PCR|Standard PCR]] for S-R-Rz/Rz1====
 +
{| class="experiments"
 +
!No.||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
 +
|}
 +
{|class="experiments"
 +
|+ PCR condition
 +
|-
 +
|94&#x2103;||2min||
 +
|-
 +
|98&#x2103;||10sec||rowspan="3"|30 cycles
 +
|-
 +
|55&#x2103;||30sec
 +
|-
 +
|68&#x2103;||4min
 +
|-
 +
|4&#x2103;||forever||
 +
|}
 +
</div>
 +
----
 +
<div class="digestion">
 +
====[[Team:Kyoto/Protocols#Restriction_Digestion|Restriction Digestion]] of <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, and PstI to check function of our Restriction Enzyme====
 +
{| class="experiments"
 +
!No.||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
 +
|}
 +
</div>
 +
----
 +
<div class="electrophoresis">
 +
====[[Team:Kyoto/Protocols#Electrophoresis|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.
 +
</div>
 +
----
 +
<div class="digestion lysis">
 +
====Digestion of the PCR products of S gene by EcoRI and SpeI and <partinfo>E0840</partinfo> by EcoRI and XbaI to insert S gene to <partinfo>E0840</partinfo>====
 +
{| class="experiments"
 +
!Name||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>||45||5||''EcoR''I 0.2||''Xba''I 0.2||0||50
 +
|}
 +
After PCR purification, evaporated them and diluted 3ul.
 +
</div>
 +
----
 +
<div class="ligation lysis">
 +
{| class="experiments"
 +
!Name||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>
</div>
</div>

Revision as of 12:42, 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

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

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

Thursday, July 22 By: Wataru

Electrophoresis of the PCR products for 40min

KyotoExp100722-1.png

Length of S and S-R-Rz/Rz1 is 370bp and 1300bp, so PCR succeeded.

Miniprep

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

The concentration of all samples was very week. Probably our shaking incubation was week.

Culture of plates and making master plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo> from 07/22 17:00 to 07/23 10:00

Friday, July 23 By: Wataru, Tomo, Makoto

Miniprep

NameConcentration(ng/µl)
<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.

Picking up number 1, 3, 5, and 7 of the products of PCR, and PCR-purification

No.NameConcentration (ng/µl)New Name
1S-R-Rz/Rz118.6-
3S77.6S1
5S-R-Rz/Rz133.6-
7S65.4S2

The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.

Retry of Standard PCR for S-R-Rz/Rz1

No.Water25mmol/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℃2min
98℃10sec30 cycles
55℃30sec
68℃4min
4℃forever

Restriction Digestion of <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, and PstI to check function of our Restriction Enzyme

No.10xBufferBSAEnzymeMilliQTotalIncubation
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

Electrophoresis of above sample for 35min

KyotoExp100723-1.png

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.

Digestion of the PCR products of S gene by EcoRI and SpeI and <partinfo>E0840</partinfo> by EcoRI and XbaI to insert S gene to <partinfo>E0840</partinfo>

Name10×BufferEnzyme 1Enzyme 2MilliQTotalIncubation
S111µl5EcoRI 0.2SpeI 0.233.650At 37℃ for 2h
S2115EcoRI 0.2SpeI 0.233.650
<partinfo>E0840</partinfo>455EcoRI 0.2XbaI 0.2050

After PCR purification, evaporated them and diluted 3ul.

NameVectorInsertLigation HighTotal
S-E08401<partinfo>E0840</partinfo> 0.5µlS1 0.512
S-E08402<partinfo>E0840</partinfo> 0.5S2 0.512
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