Team:Kyoto/Notebook

From 2010.igem.org

(Difference between revisions)
(Wednesday, July 28 By:)
(Tuesday, July 27 By: Wataru, Tomo, Kazuya, Ken, Naoi)
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!No.||Name||Length||Result
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|6||S<sub>Sam7</sub>(1)-E0840||||&#x25CE; (Use as S<sub>Sam7</sub>(1)-E0840)
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|11||S<sub>Sam7</sub>(2)-E0840||||&#x25CE; (Use as S<sub>Sam7</sub>(2)-E0840)
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Revision as of 17:05, 9 October 2010

Contents

Index

Notebook

Tuesday, July 20 By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto

Transformation

NameWellSampleCompetent CellsTotalPlateIncubationResult
<partinfo>J23100</partinfo>1-18-C1 µl2021LB (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 <partinfo>pSB4K5</partinfo> is Kanamycin-resistance, however, we plated it to LB plate (Amp+). And We didn't pre-culture <partinfo>B0015</partinfo> despite its vector is Kanamycin-resistance. So, it was predicted that we will fail the transformation of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo>.

Wednesday, July 21 By: Wataru, Ken, Makoto, Takuya Y.

Culture at 37℃ from 07/21 20:50 to 07/22 17:00 and Making Master Plate

Transformation

NameWellSampleCompetent CellsTotalPlateIncubationResult
<partinfo>pSB4K5</partinfo>1-5-G1 µl2021LB (Kan+)At 37℃, 7/21 20:50 - 7/22 16:30
<partinfo>B0015</partinfo>1-23-L12021

PCR for SRRz and S

No.WaterMgSO4dNTPs10xBufferTemplate DNAPrimer Fwd.Primer Rev. (SRRz)Primer Rev. (S)KOD Plus ver.2Total
128 µl35551.51.5-150
22835551.51.5-150
32835551.5-1.5150
42835551.5-1.5150
52835551.51.5-150
62835551.51.5-150
72835551.5-1.5150
82835551.5-1.5150
94℃2min
98℃10sec30 cycles
55℃30sec
68℃4min
4℃forever

Thursday, July 22 By: Wataru

Electrophoresis (40min) of the PCR Products

KyotoExp100722-1.png
No.NameLength(bp)Result
1SRRz1386
2SRRz1386
3S442
4S442
5SRRz1386
6SRRz1386
7S442
8S442

Marker: 100bp, 1kb, 1kb, 100bp.

Miniprep

NameConcentration
<partinfo>J23100</partinfo>18.5 (ng/µl)
<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 from 07/22 17:00 to 07/23 10:00 and Making Master Plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo>

Friday, July 23 By: Wataru, Tomo, Makoto

Miniprep

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

PCR Purification

No.NameConcentrationNew Name
1SRRz18.6 ng/µl-
3S77.6SSam7(1)
5SRRz33.6-
7S65.4SSam7(2)

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

Standard PCR for SRRz

No.WaterMgSO4dNTPs10xBufferTemplate DNAPrimer Fwd. (SRRz)Primer Rev. (SRRz)KOD plus ver.2Total
128 µl35551.51.5150
22835551.51.5150
326.54.55551.51.5150
426.54.55551.51.5150
52565551.51.5150
62565551.51.5150
94℃2min
98℃10sec30 cycles
55℃30sec
68℃4min
4℃forever

Restriction Digestion and Electrophoresis (35min) to check function of our Restriction Enzyme

No.NameSample10xBufferBSAEnzymeMilliQTotalIncubation
1<partinfo>J06702</partinfo>5 µl10.1EcoRI0.13.610At 37℃ 7/23 18:00 - 7/23 18:30
2<partinfo>J06702</partinfo>510.1XbaI0.13.610
3<partinfo>J06702</partinfo>510.1SpeI0.13.610
4<partinfo>J06702</partinfo>510.1PstI0.13.610
5<partinfo>J06702</partinfo>510.1-3.710
KyotoExp100723-1.png

Marker: 1kb.

Comparison to No. 5 (control, circular DNA), the bands of No. 1, 2, 3, and 4 was shifted. The DNA of them was linearized by Restriction enzymes. So, our restriction enzymes work correctly.

Restriction Digestion and Ligation to insert S gene to <partinfo>E0840</partinfo>

NameSample10xBufferEnzyme 1Enzyme 2MilliQTotalIncubation
SSam7(1)11 µl5EcoRI0.2SpeI0.233.650At 37℃ for 2h
SSam7(2)115EcoRI0.2SpeI0.233.650
<partinfo>E0840</partinfo>455EcoRI0.2XbaI0.2050

After PCR Purification, evaporated them and diluted 3ul.

NameVectorInsertLigation HighTotal
SSam7(1)-E0840<partinfo>E0840</partinfo>0.5µlSSam7(1)0.512
SSam7(2)-E0840<partinfo>E0840</partinfo>0.5SSam7(2)0.512

Monday, July 26 By: Wataru, Tomonori, Makoto

Electrophoresis of PCR Products

KyotoExp100726-1.png
No.NameLength(bp)Result
1SRRz1386
2SRRz1386
3SRRz1386
4SRRz1386
5SRRz1386
6SRRz1386

Marker: 1kb.

At the condition 4 (4.5µl MgSO4) and 6 (6µl MgSO4), SRRz is amplified very much. So we decided to use them.

PCR Purification

No.NameConcentrationNew Name
4SRRZ51.6 ng/µlSRRzSam7(1)
5SRRZ59.3
6SRRZ59.6SRRzSam7(2)

Transformation

NameWellSampleCompetent CellTotalPlateIncubationResult
<partinfo>E0240</partinfo>1-12-M1 µl2021LB (Amp+)At 37℃ 7/26 - 7/27×
<partinfo>I20260</partinfo>2-17-F12021LB (Kan+)×
<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

Colony PCR of SSam7-E0840 (Electrophoresis for 35min)

KyotoExp100727-1.png
No.NameLengthResult
1SSam7(1)-E08401522
2SSam7(1)-E08401522×
3SSam7(1)-E08401522
4SSam7(1)-E08401522×
5SSam7(1)-E08401522
6SSam7(1)-E08401522◎ (Use as SSam7(1)-E0840)
7SSam7(2)-E08401522×
8SSam7(2)-E08401522×
9SSam7(2)-E08401522×
10SSam7(2)-E08401522×
11SSam7(2)-E08401522◎ (Use as SSam7(2)-E0840)
12SSam7(2)-E08401522
13SSam7(2)-E08401522
+<partinfo>E0840</partinfo>1116
-None

Marker: 1kb, 100bp

Miniprep

NameConcentration
<partinfo>R0011</partinfo>26.9 ng/µl
<partinfo>B0015</partinfo>120.0
<partinfo>E0840</partinfo>120.1

Restriction Digestion

Sample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotalIncubation
<partinfo>B0015</partinfo>30 µl50.5EcoRI0.4XbaI0.313.750At 37℃ 16:45 - 18:00
SRRzSam7(1)4050.5EcoRI0.4SpeI0.43.850
SRRzSam7(2)4050.5EcoRI0.4SpeI0.43.850

Transformation

NameSampleCompetent CellsTotalPlateIncubationResult
SRRzSam7(1)-B0015
SRRzSam7(2)-B0015

Wednesday, July 28 By:

Miniprep

NameConcentration
SSam7(1)-E084095.5 ng/%micro;l
SSam7(2)-E084098.6

Diluted SSam7(1)-E0840 and SSam7(2)-E0840 20 times with water, and used as template DNA.

Deletion PCR to delete a functional domain of S gene

WaterMgSO4dNTPs10xBufferPrimer Fwd.Primer Rev.Template SSam7(1)-E0840Template SSam7(2)-E0840KOD Plus ver.2Total
SSam7,ΔTMD1(1)-E0840 (1)283551.51.55-150
SSam7,ΔTMD1(1)-E0840 (2)283551.51.55-150
SSam7,ΔTMD1(2)-E0840 (1)283551.51.5-5150
94℃2min
98℃10sec35 cycles
55℃30sec
68℃4min
4℃forever

Restriction Digestion to check the function of DpnI

NameSamplefast digestion bufferDpnIMilliQTotal
SSam7(1)-E0840310.15.810
SSam7(2)-E0840310.15.810

Electrophoresis for 35min

KyotoExp100728-1.png
No.NameLengthResult
1Not digested SSam7(1)-E0840
2Not digested SSam7(2)-E0840
3Digested SSam7(1)-E0840
4Digested SSam7(2)-E0840

Marker: 1kb, 100bp

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

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]pSB4K5(E-P)1R0011(E-S)1E02401(X-P)131517:30 - 20:20
R0011-E02402[Low]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]pSB4K5(E-P)1J23101-E0240(E-P)12420:00-20:30

Transformation

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

Thursday, August 5 By:

Result of Transformation

R0011-E02401[Low]Many colonies
R0011-E02402[Low]
J23101-E0240[Low]

pSB4K5 is inserted RFP generator. We didn't distinguish this inserted parts from low copy plasmid backbone, so self-ligated colony is red. So, white colony is correctly inserted parts.

However, white colonies and green colonies are observed in R0011-E02401[Low] and R0011-E02402[Low] plate. We cultured both white and green colonies.

In J23101-E0240[Low], Many of colonies are red, but green colonies are observed. We cultured green colonies.

NameColorIncubation
R0011-E02401[Low]-1Green Colony8/5-8/6
R0011-E02401[Low]-2Green Colony
R0011-E02401[Low]-3White Colony
R0011-E02401[Low]-4White Colony
R0011-E02402[Low]-1Green Colony
R0011-E02402[Low]-2White Colony
R0011-E02402[Low]-3White Colony
R0011-E02402[Low]-4White Colony
J23101-E0240[Low]-1Green Colony
J23101-E0240[Low]-2Green Colony
J23101-E0240[Low]-3Green Colony
NameConcentration(ng/µL)
SΔTMD1-E08401-1-A28.9
SΔTMD1-E08401-1-B25.3
SΔTMD1-E08402-A26.6
SΔTMD1-E08402-B24.0

As a result, deletion is succeeded, however, point mutation is failed. It is because DpnI is too little to digest all of template DNA.


Friday, August 6

Miniprep

Name
R0011-E02401[Low]-1
R0011-E02401[Low]-2
R0011-E02401[Low]-3
R0011-E02401[Low]-4
R0011-E02402[Low]-1
R0011-E02402[Low]-2
R0011-E02402[Low]-3
R0011-E02402[Low]-4
J23101-E0240[Low]-1
J23101-E0240[Low]-2
J23101-E0240[Low]-3

Restriction Digestion

NameSample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
R0011-E02401[Low]-15060.6EcoRI0.3PstI0.32.860
R0011-E02401[Low]-25060.6EcoRI0.3PstI0.32.860
R0011-E02401[Low]-35060.6EcoRI0.3PstI0.32.860
R0011-E02401[Low]-45060.6EcoRI0.3PstI0.32.860
R0011-E02402[Low]-15060.6EcoRI0.3PstI0.32.860
R0011-E02402[Low]-25060.6EcoRI0.3PstI0.32.860
R0011-E02402[Low]-35060.6EcoRI0.3PstI0.32.860
R0011-E02402[Low]-45060.6EcoRI0.3PstI0.32.860
J23101-E0240[Low]-15060.6EcoRI0.3PstI0.32.860
J23101-E0240[Low]-25060.6EcoRI0.3PstI0.32.860
J23101-E0240[Low]-35060.6EcoRI0.3PstI0.32.860
Electrophoresis
M100bpcolspan="4"colspan="4"|
Mλ
Mλ
M100bp
1J23101-E0240[Low]-1
2J23101-E0240[Low]-2
3J23101-E0240[Low]-1
4R0011-E02401[Low]-1
5R0011-E02401[Low]-2
6R0011-E02401[Low]-3}
7R0011-E02401[Low]-4}
8R0011-E02402[Low]-1
9R0011-E02402[Low]-2}
10R0011-E02402[Low]-3}
11R0011-E02402[Low]-4}
12J23101-E0240[Low]-1
13J23101-E0240[Low]-2

KyotoExp100806-1.png White colonies are not inserted <partinfo>R0011</partinfo> but its vector. Top10 we used are deleted Lac operon. Then, correctly inserted parts is green because of the lack of lacI gene.

Error PCR (Retry)

NameWater25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2Primer VF2(10µM)Primer VR(10µM)Template ΔTMD failed(50ng/µL)KOD plus ver.2Total
ΔTMD①323551.51.51150
ΔTMD②323551.51.51150
94℃2min
98℃10sec25 cycles
68℃4min
Add DpnI 2µl
Incubate1h
4℃forever

Transformation

NameConc(/µL)Sample Volum(µL)Competent Cell(µL)TotalPlateIncubation
ΔTMD①-45054LB kan8/6~8/9
ΔTMD②-45054
2-17-F-25052
2-I-525052LB amp


Monday, August 9 By: Wataru, Tomonori, Ken, Takuya

Miniprep of MS and ML

Sample numberconcentration(ng/µL)
MS116.2
ML146.6

Transfotrmation of MS and ML

Sampleconc(ng/µL)Sample vol(µL)Competent CellCompetent cell vol(µL)Total vol(µL)PlateIncuvation
MS116.22KRX5052LB kanamycin8/9 18:00‾8/10 12:00
ML146.62KRX5052

Restriction enzyme digestion and ethanol precipitation

To use lac p for next ligation, we digested 1-6-G by EroRI and PstI

Sample10x BufferBSAEnzyme (EcoRI)Enzyme (PstI)MilliQTotal
5060.60.50.52.460

Incubate 37℃ 8/9 16:20‾18:20

After restriction enzyme digestion, we did ethanol precipitation.

Ligation and Transformation

SampleConc (nu/µL)Sample vol (µL)Competent cellCompetent cell vol (µL)Total vol (µL)PlateIncuvation
Lac p (low)-2KRX5052LB kanamycin8/9 20:00‾8/10 9:00
2C25052


===Tuesday, August 10 By: Wataru, Tomonori, Ken, Fumitaka

Making culture plate on lac p (low), MS and ML

Lac p (low)KRXMany colonies
C2
MSKRX
MLKRX

Minprep of ΔTMD1+GFP

Sample numberConcentration (ng/µL)
1-19.9
1-227.3
2-143.2
2-234.7

37℃ 8/9 18:00‾8/10 9:00

Culture and Master Plate


===Wednesday, August 11 By: Wataru, Naoi, Ken, Takuya

SampleMediumCloudIncubation
1Kanamycino37℃8/10 20:00‾8/11 9:00
Ampicillinx
2Kanamycino
Ampicillino
3Kanamycino
Ampicillinx
4Kanamycino
Ampicillinx
5Kanamycino
Ampicillinx
6Kanamycino
Ampicillino
7Kanamycino
Ampicillinx

Discussion: About sample 1, 3, 4, 5 and 7, lac promoter was correctly inserted in low copy plasmid. About sample 2 and 6, low copy plasmid and vector derived from lac promoter were ligated. We decided to use sample 1 or 3.

Miniprep of C2+lac(low), S-R-Rz 1', 3'

lac(low)1 : 31.2 (ng/µL) lac(low)2 : 29.9 (ng/µL)

Restriction Digestion and electrophoresis of lac (low) 1 and 3

NameEcoRIPstI
10.2-
2-0.2
30.20.2
N--

Sample: 1-1, 1-2, 1-3, 1-N, 3-1, 3-2, 3-3, 3-N

M 1-1 1-2 1-3 1-N M M 3-1 3-2 3-3 3-N M

KyotoExp100811-1.png

Discussion: Each enzyme correctly cut samples.

Screening PCR of SRRz

Sample: 1-20

Control: P(1-23L) P'(2-8E) N

Maker: lambda

M N P P' P 1 2 3 4 5 6 M

KyotoExp100811-2.png

7 8 9 10 11 12 13 M 14 15 16 18 19 20 M

KyotoExp100811-3.png

Discussion: All of the sample were self-ligation of DT and SRRz weren't inserted.


Thursday, August 12 By: Wataru, Ken

Restriction Digestion and electrophoresis of <partinfo>B0015</partinfo>

Sample nameTemplate10xbuffer100xbufferEcoRIXbaI 1XbaI 2SpeIPstI 1PstI 2WaterTotal
1310.10.2-----5.710
2310.1-0.2----5.710
3310.1--0.2---5.710
4310.1---0.2--5.710
5310.1----0.2-5.710
6310.1-----0.25.710
N310.1------5.910

Sample: 1-6, N Maker: lambda, 100

M 1 2 3 4 5 6 N M M M

KyotoExp100812-1.png

Discussion: Each enzyme correctly cut each sample and was active.


===Thursday, August 19 By: Wataru, Tomo, Ken

Miniprep of SΔTMD1GFP

29.6(ng/µg)

Point mutation PCR of ΔTMD1GFP

Sample numberTemplate10xbufferdNTPsMgSO4Primer 1Primer 2WaterKOD-plus-Total
11.55531.51.531.5150
21.55531.51.531.5150
control1.55531.51.532.5-50
94(℃)2min
9810sec30cycles
5530sec
683.5min
4.0forever

Restriction Digestion(DpnI): 17:50-18:50

Electrophoresis

Sample: 1, 2, Control Marker: lambda, 100 KyotoExp100819-1.png

Ligation and Transformation

We named point mutation PCR products rΔTMD1GFP.


Monday, August 23 By: Wataru, Tomo, Ken, Fumitaka, Tasuku

Miniprep of ΔTMD1

Sample numberConcentration(ng/µg)
1-158.9
2-249.9

Sequencing of ΔTMD1 and MS

Sample: rδTMD1GFP1-1, 2-2, and MS

Discussion: The sequencing was in success and the results were desirable. It meant point mutation of δTMD1GFP was succeeded and sequence of MS was confirmed. We decided to use rδTMD1GFP.

Screening PCR of SRRz-DT

Sample: 1-13, Marker: lambda and 100, Control:P(1-23L) and N

90℃10min
94℃30sec35cycles
50℃30sec
72℃1.5min
72℃4min
4℃hold

M 1 2 3 4 5 6 7 8 9 10 11 12 13 P N M

KyotoExp100823-1.png

Discussion: We found the band; about 200bp, and it meant the lligation was completed successfully.

Deletion PCR of rΔTMD1GFP 2-2

Sample10xdNTPsPrimer1Primer2TemplateWaterKOD-plus-Total
1551.51.5135150
2551.51.5135150
Control551.51.5135-50
94℃2min
94℃10sec35cycles
56℃30sec
68℃3.5min
4℃hold

Restriction Digestion(DpnI)

Template25(µL)
DpnI1
Total26

19:10-20:10

Ligation

SampleTemplateWaterLigation highT4 Kinasetotal
1365115
2365115
Control365115

20:15-21:15

Transformation

We named sample 1, 2 and control rrδTMD1GFP1, 2 and control.


Tuesday, August 24 By:Ken, Tomo, Tasuku, Takuya

Retry of deletion PCR of rδTMD1 GFP

Sample10xdNTPsMgSO4Primer1Primer2TemplateWaterKOD-plus-Total
15531.51.5132150
25531.51.5132150

Control||5||5||3||1.5||1.5||1||32||1||50

94℃2min
94℃10sec35cycles
58℃30sec
68℃3.5min
4℃hold

Restriction Digestion (DpnI)

14:15-15:15

Electrophoreis

Sample: 1, 2, and control, Maker: 100 and lambda M 1 2 C M

KyotoExp100824-1.png

We found the band of sample 1 and 2 about 3000bp and there wasn't the band of sample control. So, we confirmed the PCR and RE were completed successfully.

Ligation

Point mutation of SRRz

Sample10xdNTPsMgSO4Primer1Primer2TemplateWaterKOD-plus-total
15531.51.5132150
25531.51.5132150
control5531.51.5132150
94℃2min
98℃10sec30cycles
55℃30sec
68℃4min
4℃hold

Restriction Digestion(DpnI), electrophoresis and ligation

KyotoExp100824-2.png

We could find point mutation PCR and restriction enzyme of DpnI was done.

=PCR of E0240

Sample10}dNTPsMgSO4VF2VRTemplateWaterKOD-plus-Total
15531.51.5131.5150
25531.51.5131.5150

=PCR Purification

Sample1: 5.5*50(ng/µL) Sample2: 5.2*50(ng/µL)

Restriction Digestion(EcoRI, PstI) and Gel extraction

Sample1: 28.8 (ng/µL) Sample2: 26.4 (ng/µL)

Transformation

Sample: rrΔTMD1GFP1. 2. control, and rSRRz1. 2. control


Wednesday, August 25 By:Ken, Tomo, Kazuya, Tasuku, Takuya

Making culture and Master plate

rrΔTMD1-1Many Colonies
rrΔTMD1-2
rrΔTMD1-C-zero
rSRRz-1Many Colonies
rSRRz-2
rSRRz-C-zero

Miniprep of 1-5G

29.0 (ng/µL)

Restriction Digestion and purification of 1-5G(low copy plasmid) and lac low

Sample nameTemplate10xbuffer100xbufferEcoRISpeIPstIWaterTotal
1-5G5060.60.40.4-2.660
Lac low1040.4-0.30.32540
Sample NameConcentration(ng/µL)
1-5G18.4
Lac low8.6

Ligation of <partinfo>E0240</partinfo> and <partinfo>pSB4K5</partinfo>, Transformation


===Thursday, August 26 By:Ken, Tomo, Kazuya, Tasuku, Takuya, Fumitaka

Miniprep

Sample nameConcentration(ng/µL)
constP(0.7)44.5

Restriction Digestion of constP(0.7)

Template10xbuffer100xbufferSpeIPstIWaterTotal
2540.40.30.31040

Purification of constP (0.7)

49.8 ng/µL


Friday, August 27 By:Ken, Tomo, Kazuya, Fumitaka

Making master plate of E0240 low

Sample NameConcentration(ng/µL)
rrΔTMD1 1-220.9
rSRRz 1-116.4

Restriction Digestion of rrΔTMD1 and rSRRz

Sample nameTemplate10xbuffer100xbufferXbaIPstIWaterTotal
rrΔTMD1 1-24560.60.30.37.860
rSRRz 1-14560.60.30.37.860

(13:20-14:20)

Purification

rrΔTMD1 1-244.7
rSRRz 1-156.1

Lagation and transformation

lacP + rrΔTMD1 1-2 constP (0.7) + rrΔTMD1 1-2 lac low + rSRRz 1-1


Monday, August 30 By: Tomonori, Kazuya, Tasuku, Ken

Making culture and Master plate

lacP rrΔTMD1GFPMany colonies
lacP rrΔTMD1GFP(control)Some colonies
constP rrΔTMD1GFPMany colonies
constP rrΔTMD1GFP(control)Many colonies
lacP rSRRz lowNo colony
lacP rSRRz low(control)No colony

Discussion: There ware some colonies, which emitted green light, on the plate 1. So, we cultured those colonies on master plate. On the plate 5 and 6, even though we used KRX, which is able to repress lac promoter, colonies might be dead. However, we still have to do some experience so that we confirm lac promoter cannot repress enough and E. coli cannot survive.


===Tuesday, August 31 By: Tomonori, Takuya Y., Kazuya, Tasuku, Takuya, Ken

Miniprep

constP (0.3)48.5 (ng/µL)
lac rrΔTMD1107.3

RE of constP (0.3) and lac rrΔTMD1

Gel Extraction of lac rrΔTMD1

File:KyotoExp100831-1.png

45min

Discussion: There were two band at the bottom of the gel. It was too long -45min-, and insert and vector might be contaminated. But we went on next operation.

Purification of constP (0.3) and lac rrΔTMD1

constP (0.3)5.8 (ng/µL)
lac rrΔTMD17.8 (ng/µL)

Ligation and transformation

InsertVector
lac rrΔTMD1constP (0.3)


===Wednesday, September 1 By: Tomonori, Kazuya, Tasuku, Fumitaka, Ken

Making culture and Master plate

lac rrΔTMD1 constPmany colonies
lac rrΔTMD1 const (control)many colonies

Screenig PCR of lacP-rrΔTMD1GFP-constP Sample: 1-13 Control: Positive (1-23L) Maker: lambda, 100

   M  1  2  3  4   5  6  7     8  9  10 11 12  13  P M

File:KyotoExp100901.png

Discussion: All of the sample except sample 10 might be self-ligation products of constP.

Miniprep

rSRRz 1-133.8 (ng/µL)
low56.0 (ng/µL)

Restriction Digestion of rSRRz and low

Sample nameTemplate10xbuffer100xbufferEcoRIPstIWaterTotal
rSRRz2040.40.30.31540
low2040.40.30.31540

(13:25-14:30)

Purification

rSRRz6.5 (ng/µL)
low16.8

Ligation and transformation

Insert: rSRRz 1-1 Vector: low copy plasmid


Thursday, September 2 By: Tomonori, Tomo, Takuya, Ken

Making culture and Master plate

rSRRz low13 colonies
rSRRz low (Control)13colonies

Screening PCR of rSRRz low

Sample: rSRRz (1-13) Maker: lambda, 100 Control: Positive (1-23L), Neganive

M 1 2 3 4 5 6 7 8 9 10 11 12 13 P N M

File:KyotoExp100902.png

Discussion: From sample 1, two vectors might be ligated. Sample 3 and 4, rSRRz might be inserted in low copy plasmid correctly. Sample 11, it might be the self-ligation product of low copy plasmid. Anyway, we decided to culture those 4 colonies on master plate.

Friday, September 3 By: Tomonori, Tomo, Kazuya, Tasuku, Fumitaka, Ken

Making culture

lac rrΔTMD1 1, 3 rrΔTMD1 1-1, 1-2 rSRRz 1-1, 1-2 ML