Team:Kyoto/Notebook

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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
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.

Ligation

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


Monday, July 26 By: Wataru, Tomonori, Makoto

Electrophoresis of PCR Products

KyotoExp100726-1.png

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.

PCR Purification

No.Concentration (ng/µl)New Name
451.6SRRz1
559.3
659.6SRRz2

Transformation

NameWellSample (µl)Competent Cell (µl)Total (µl)PlateIncubationResult
<partinfo>E0240</partinfo>1-12-M12021LB (Amplicillin+)At 37℃ 7/26 - 7/27×
<partinfo>I20260</partinfo>2-17-F12021LB (Kanamycin+)×
<partinfo>J04450</partinfo>1-5-E12021×

Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>

Tuesday, July 27 By: Wataru, Tomo, Kazuya, Ken, Naoi

Team:Kyoto/Protocols#Colony_PCR~Colony PCR of S-E0840 (Electrophoresis for 35min)

Marker12345678910111213+-Marker
1kbS-E08401S-E08402E0840None100bp
KyotoExp100727-1.png

As a result, 1, 3, 5, 6, 11, 12, and 13 are inserted S gene correctly. So, we decided to use 6 as S-E08401 and 11 as S-E08402.

Miniprep

NameConcentration(ng/µL)
<partinfo>R0011</partinfo>26.9
<partinfo>B0015</partinfo>120.0
<partinfo>E0840</partinfo>120.1

Restriction Digestion

Sample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotalIncubation
<partinfo>B0015</partinfo>3050.5EcoRI0.4XbaI0.313.750At 37℃ 16:45 - 18:00
SRRz14050.50.40.43.850
SRRz24050.50.40.43.850

Ligation

Transformation

NameSample (µl)Competent Cells (µl)Total (µl)PlateIncubationResult
SRRz1-B0015rowspan="2"rowspan="2"○
SRRz2-B0015○


Wednesday, July 28 By:

==Miniprep

NameConcentration(ng/µl)
S-E0840195.5
S-E0840298.6

Diluted S-E08401 and S-E08402 20 times with water, and used as template DNA.

Deletion_PCR to delete a functional domain of S gene

Water25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2Primer Forward(10µM)Primer Reverse(10µM)Template S-E08401Template S-E08402KOD Plus ver.2Total
SΔTMD1-E08401-1283551.51.55-150
SΔTMD1-E08401-2283551.51.55-150
SΔTMD1-E08402283551.51.5-5150
94℃2min
98℃10sec35 cycles
55℃30sec
68℃4min
4℃forever

Restriction Digestion to check the function of DpnI

NameSamplefast digestion bufferDpnIMilliQTotal
S-E08401310.15.810
S-E08402310.15.810

Electrophoresis for 35min

Marker1234Marker
1kbNot digested S-E08401Not digested S-E08402Digested S-E08401Digested S-E08402100bp
KyotoExp100728-1.png

DpnI works correctly.


Thursday, July 29 By:

Restriction Digestion

NameSample volumeFastdigestion BufferEnzyme 1MilliQTotalIncubation
SΔTMD1-E08401-1506DpnI0.23.86007/29 09:40 - 07/29 11:00
SΔTMD1-E08402506DpnI0.23.860

Ligation and Pospholylation

NameSampleMilliQLigation HighT4 KinaseTotalIncubation
SΔTMD1-E08401-127511507/29 11:30 ~ 07/29 13:00
SΔTMD1-E08402275115

Transformation

NameSample Volume(µL)Competent Cell(µL)TotalPlateIncubationResult
SΔTMD1-E08401-133033LB Amp+07/29 ~ 07/30○
SΔTMD1-E0840233033○


Monday, August 2 By: Wataru, Ken

Miniprep

NameConcentration(ng/µL)
SΔTMD1-E0840-152.7
SΔTMD1-E0840-254.4
SΔTMD1-E0840-389.5
<partinfo>pSB4K5</partinfo>50.7
<partinfo>R0011</partinfo>18.6

Standard PCR of <partinfo>E0240</partinfo>

E240 is very important parts to measure RPU of promoters in iGEM. However, we failed to transfect it to E.coli from parts kit of iGEM. So we decided to amplify this parts by PCR.

NameWater25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2Primer VF2(10µM)Primer VR(10µM)Template E240KOD Pllus ver.2Total
E02401283551.51.55150
E02402283551.51.55150
94℃2min
98℃10sec35 cycles
55℃30sec
68℃4min
4℃forever

Electrophoresis

PCR Purification

Sample numberConcentration(ng/µL)
E0240142.6
E0240255.3

Restriction Digestion for inserting <partinfo>E0240</partinfo> to pSB4K5 by 3A assembly

NameSample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
E02401(X-P)3050.5XbaI0.2PstI0.214.150
E02402(X-P)3050.5XbaI0.2PstI0.214.150

PCR Purification

NameConcentration(ng/µL)Volume(µL)
E02401(X-P)21.840
E02402(X-P)32.445

Stored at -20℃.

Error PCR

NameWater25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2Primer VF2(10µM)Primer VR(10µM)Template Δ1TemplateTemplateKOD Pllus ver.2Total
SΔTMD1-E08401-1323551.51.51--150
SΔTMD1-E08401-2323551.51.5-1-150
SΔTMD1-E08402323551.51.5--1150
94℃2min
98℃10sec20 cycles
68℃4min
4℃forever

Transformation

NameSample (µl)Competent Cells (µl)Total (µl)PlateIncubationResult
SΔTMD1-E08401-122022rowspan="3"rowspan="3"○
SΔTMD1-E08401-222022}
SΔTMD1-E0840222022○


Tuesday, August 3 By:

=Culture of each two colonies of SΔTMD1-E08401-1 and SΔTMD1-E08402 for 37℃ 08/03-08/04

Miniprep for Construction of Measure(lacP) and Measure(Standard)

Sample numberConcentration(ng/µL)
<partinfo>pSB4K5</partinfo>60.7
<partinfo>R0011</partinfo>26.8

Restriction Digestion

NameSample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
R00115060.6EcoRI0.2SpeI0.2360
pSB4K5(E-P)5060.6EcoRI0.2PstI0.2360
E02401(X-P)5060.6XbaI0.2PstI0.2360
E02402(X-P)5060.6XbaI0.2PstI0.2360

PCR Purification

Sample numberConcentration(ng/µL)
pSB4K5(E-P)39.5
E02401(X-P)21.8
E02402(X-P)32.4

pSB4K5(E-P) is concentrated 10µL and E02401(X-P), E02402(X-P) are concentrated 1µL.

Ethanol Precipitation

Dilution of <partinfo>pSB4K5</partinfo> by 2µl MilliQ

Ligation

VectorInsert 1Insert 2Ligation HighTotalIncubation
R0011-E02401[Low Copy]pSB4K5(E-P)1R0011(E-S)1E02401(X-P)131517:30 - 20:20
R0011-E02402[Low Copy]pSB4K5(E-P)1R0011(E-S)1E02402(X-P)1315

Standard PCR of <partinfo>J23101</partinfo>-<partinfo>E0240</partinfo> that is important in the measurement of RPU

NameWater25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2Primer VF2(10µM)Primer VR(10µM)Template J23101-E0240KOD plus ver.2 Total
J23101-E02401323551.51.51150
J23101-E02402323551.51.5-150
94℃2min
98℃10sec30 cycles
55℃30sec
68℃4min
4℃forever

PCR Purification

NameConcentration(ng/µL)
J23101-E024040.6

Restriction Digestion

NameSample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
J23101-E0240(E-P)4560.6EcoRI0.2PstI0.2860

PCR Purification

NameConcentration(ng/µL)Volume(µL)
J23101-E0240(E-P)74.130

J23101-E0240(E-P) is concentrated 7µL

Ligation

VectorInsertLigation HighTotalIncubation
J23101-E0240[Low Copy]pSB4K5(E-P)1J23101-E0240(E-P)12420:00-20:30

Transformation

NameConc(/µL)Sample Volume(µL)Competent Cell(µL)TotalPlateIncubation
R0011-E02401[Low Copy]-12021LB kan8/3~8/4
R0011-E02402[Low Copy]-12021
J23101-E0240[Low Copy]-12021

</div>

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