Team:Kyoto/Notebook

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====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>====
====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"
{| class="experiments"
-
!Name||10×Buffer||Enzyme 1||Enzyme 2||MilliQ||Total||Incubation
+
!Name||Sample Volume (&micro;l)||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>1</sub>||11||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
|S<sub>2</sub>||11||5||''EcoR''I 0.2||''Spe''I 0.2||33.6||50

Revision as of 12:43, 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>

NameSample Volume (µl)10×BufferEnzyme 1Enzyme 2MilliQTotalIncubation
S1115EcoRI 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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