Team:Kyoto/Notebook1

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|I20260 [pSB4K5] (3) [EP]||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
|I20260 [pSB4K5] (3) [EP]||50||6||0.6||EcoRI||0.3||PstI||0.3||2.8||60
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========Electrophoresis======
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=====Electrophoresis=====
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[[Image:KyotoExp100806-1.png|300px|right]]
[[Image:KyotoExp100806-1.png|300px|right]]
White colonies are not inserted R0011 but its vector. Top10 we used are deleted Lac operon. Then, correctly inserted parts is green because of the lack of ''lac''I gene.
White colonies are not inserted R0011 but its vector. Top10 we used are deleted Lac operon. Then, correctly inserted parts is green because of the lack of ''lac''I gene.
 +
=====Error PCR (Retry)=====
=====Error PCR (Retry)=====
{| class="experiments"
{| class="experiments"

Revision as of 14:09, 23 October 2010

Contents

Notebook1

Construction

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

Transformation
NameWellSampleCompetent CellsTotalPlateIncubationColony
J231001-18-C1 µL2021LB (Amp+)At 37℃, 7/20 20:50 - 7/21 17:00
J231051-18-M12021
J231161-20-M12021
R00111-6-G12021
E08401-12-O12021
J067022-8-E12021
pSB4K51-5-G12021×
B00151-23-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 at 37℃ from 07/21 20:50 to 07/22 17:00 and Making Master Plate
Transformation
NameWellSampleCompetent CellsTotalPlateIncubationColony
pSB4K51-5-G1 µL2021LB (Kan+)At 37℃, 7/21 20:50 - 7/22 16:30
B00151-23-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℃10s30 cycles
55℃30s
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
J2310018.5 (ng/µL)
J2310512.5
J2311614.6
R00118.6
E084012.1
J0670214.7

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 pSB4K5 and B0015

Friday, July 23 By: Wataru, Tomo, Makoto

Miniprep
NameConcentration
pSB4K579.2 (ng/µL)
B0015-

We lost B0015 by our mistake. The concentration of pSB4K5 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.

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℃10s30 cycles
55℃30s
68℃4min
4℃forever
Restriction Digestion and Electrophoresis (35min) to check function of our Restriction Enzyme
No.NameSample10xBufferBSAEnzymeMilliQTotalIncubation
1J067025 µL10.1EcoRI0.13.610At 37℃ 7/23 18:00 - 7/23 18:30
2J06702510.1XbaI0.13.610
3J06702510.1SpeI0.13.610
4J06702510.1PstI0.13.610
5J06702510.1-3.710
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 E0840
NameSample10xBufferEnzyme 1Enzyme 2MilliQTotalIncubation
SSam7(1)11 µL5EcoRI0.2SpeI0.233.650At 37℃ for 2h
SSam7(2)115EcoRI0.2SpeI0.233.650
E0840455EcoRI0.2XbaI0.2050

After PCR Purification, evaporated them and diluted 3µL.

NameVectorInsertLigation HighTotal
SSam7(1)-E0840E08400.5µLSSam7(1)0.512
SSam7(2)-E0840E08400.5SSam7(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 CellTotalPlateIncubationColony
E02401-12-M1 µL2021LB (Amp+)At 37℃ 7/26 - 7/27×
I202602-17-F12021LB (Kan+)×
J044501-5-E12021×
Culture of pSB4K5, E0840, and B0015

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
+E08401116
-None

Marker: 1kb, 100bp

Miniprep
NameConcentration
R001126.9 ng/µL
B0015120.0
E0840120.1
Restriction Digestion
NameSample2 bufferBSAEnzyme 1Enzyme 2MilliQTotalIncubation
B001530 µL50.5EcoRI0.4XbaI0.313.750At 37℃ 16:45 - 18:00
SRRzSam7(1)4050.5EcoRI0.4SpeI0.43.850
SRRzSam7(2)4050.5EcoRI0.4SpeI0.43.850
Ligation
Transformation
NameSampleCompetent CellsTotalPlateIncubationColony
SRRzSam7(1)-B0015
SRRzSam7(2)-B0015


Wednesday, July 28 By:

Miniprep
NameConcentration
SSam7(1)-E084095.5 ng/µL
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 (1)Template (2)KOD Plus ver.2Total
SSam7,ΔTMD1(1)-E0840 (1)28 µL3551.51.55-150
SSam7,ΔTMD1(1)-E0840 (2)283551.51.55-150
SSam7,ΔTMD1(2)-E0840 (1)283551.51.5-5150
SSam7,ΔTMD1(2)-E0840 (2)283551.51.5-5150
94℃2min
98℃10s35 cycles
55℃30s
68℃4min
4℃forever
Restriction Digestion to check the function of DpnI
NameSamplefast digestion bufferDpnIMilliQTotal
SSam7(1)-E08403 µL10.15.810
SSam7(2)-E0840310.15.810
Electrophoresis for 35min
KyotoExp100728-1.png
No.NameLengthResult
1Not digested SSam7(1)-E08403363bp
2Not digested SSam7(2)-E08403363
3Digested SSam7(1)-E08401021, 933, 402, 341, 258, 105, ...
4Digested SSam7(2)-E08401021, 933, 402, 341, 258, 105, ...

Marker: 1kb, 100bp DpnI works correctly.


Thursday, July 29 By:

Restriction Digestion
NameSample volumeFastdigestion BufferEnzyme 1MilliQTotalIncubation
SSam7,ΔTMD1(1)-E0840 (1)50 µL6DpnI0.23.86007/29 09:40 - 07/29 11:00
SSam7,ΔTMD1(2)-E0840 (1)506DpnI0.23.860
Ligation and Phosphorylation
NameSampleMilliQLigation HighT4 KinaseTotalIncubation
SSam7,ΔTMD1(1)-E0840 (1)2 µL7511507/29 11:30 ~ 07/29 13:00
SSam7,ΔTMD1(2)-E0840 (1)275115
Transformation
NameSample VolumeCompetent CellTotalPlateIncubationColony
SSam7,ΔTMD1(1)-E0840 (1)3 µL3033LB (Amp+)07/29 ~ 07/30
SSam7,ΔTMD1(2)-E0840 (1)33033


Monday, August 2 By: Wataru, Ken

Miniprep
NameConcentration
SSam7,ΔTMD1-E0840 (1)52.7 ng/µL
SSam7,ΔTMD1-E0840 (2)54.4
SSam7,ΔTMD1-E0840 (3)89.5
pSB4K550.7
R001118.6
PCR of E0240

E0240 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.

NameWaterMgSO4dNTPs10xBufferPrimer VF2Primer VRTemplate E0240KOD Pllus ver.2Total
E0240(1)28 µL3551.51.55150
E0240(2)283551.51.55150
94℃2min
98℃10s35 cycles
55℃30s
68℃4min
4℃forever
Electrophoresis
PCR Purification
NameConcentration
E0240(1)42.6 ng/µL
E0240(2)55.3
Restriction Digestion for inserting E0240 to pSB4K5 by 3A assembly
NameSample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
E0240(1) [XP]30 µL50.5XbaI0.2PstI0.214.150
E0240(2) [XP]3050.5XbaI0.2PstI0.214.150
PCR Purification
NameConcentrationVolume
E0240(1) [XP]21.8 ng/µL40 µL
E0240(2) [XP]32.445

Stored at -20℃.

Error PCR
NameWaterMgSO4dNTPs10xBufferPrimer VF2Primer VRTemplate (1)Template (2)Template (3)KOD Pllus ver.2Total
SSam7,ΔTMD1-E0840 (1)32 µL3551.51.51--150
SSam7,ΔTMD1-E0840 (2)323551.51.5-1-150
SSam7,ΔTMD1-E0840 (3)323551.51.5--1150
94℃2min
98℃10s20 cycles
68℃4min
4℃forever
Restriction Digestion of SSam7,ΔTMD1-E0840 by DpnI
Transformation
NameSampleCompetent CellsTotalPlateIncubationColony
SSam7,ΔTMD1-E0840 (1)2 µL2022--
SSam7,ΔTMD1-E0840 (2)22022×
SSam7,ΔTMD1-E0840 (3)22022

Tuesday, August 3 By:

Culture

Picked two colonies from SSam7,ΔTMD1-E0840 (1), and SSam7,ΔTMD1-E0840 (3), and cultured at 37℃ from 08/03 to 08/04.

Miniprep
NameConcentration
pSB4K560.7 ng/µL
R001126.8
Restriction Digestion
NameSample2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
R0011 [ES]50 µL60.6EcoRI0.2SpeI0.2360
pSB4K5 [EP]5060.6EcoRI0.2PstI0.2360
E0240(1) [XP]5060.6XbaI0.2PstI0.2360
E0240(2) [XP]5060.6XbaI0.2PstI0.2360
PCR Purification
NameConcentration
pSB4K5 [EP]39.5 ng/µL
E0240(1) [XP]21.8
E0240(2) [XP]32.4

pSB4K5 [EP] is concentrated 10µL and E0240(1) [XP], E0240(2) [XP] are concentrated 1µL.

Ethanol Precipitation

After ethanol precipitation, we diluted pSB4K5 by 2µL MilliQ

Ligation
NameVectorInsert 1Insert 2Ligation HighT4 KinaseTotalIncubation
R0011-E0240(1) [pSB4K5]pSB4K5 [EP]1R0011 [ES]1E0240(1) [XP]131517:30 - 20:20
R0011-E0240(2) [pSB4K5]pSB4K5 [EP]1R0011 [ES]1E0240(2) [XP]1315
PCR of I20260
NameWaterMgSO4dNTPs10xBufferPrimer VF2Primer VRTemplate I20260KOD plus ver.2Total
I20260 (1)32µL3551.51.51150
I20260 (2)323551.51.5-150
94℃2min
98℃10s30 cycles
55℃30s
68℃4min
4℃forever
PCR Purification
NameConcentration
I2026040.6 ng/µL
Restriction Digestion
NameSample volume2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
I20260 [EP]45 µL60.6EcoRI0.2PstI0.2860
PCR Purification
NameConcentrationVolume
I20260 [EP]74.1 ng/µL30

I20260 [EP] is concentrated at 7µL

Ligation
VectorInsertLigation HighTotalIncubation
I20260 [pSB4K5]pSB4K5 [EP]1I20260 [EP]12420:00-20:30
Transformation
NameSampleCompetent CellTotalPlateIncubationColony
R0011-E0240(1) [pSB4K5]1 µL2021LB (Kan+)08/03-08/04
R0011-E0240(2) [pSB4K5]12021
I20260 [pSB4K5]12021


Thursday, August 5 By:

Culture and Master Plates

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-E0240(1) [pSB4K5] and R0011-E0240(2) [pSB4K5] plate. We cultured both white and green colonies.

In I20260 [pSB4K5], Many of colonies are red, but green colonies are observed. We cultured green colonies.

NameColorIncubation
R0011-E0240(1) [pSB4K5] (1)Green Colony8/5-8/6
R0011-E0240(1) [pSB4K5] (2)Green Colony
R0011-E0240(1) [pSB4K5] (3)White Colony
R0011-E0240(1) [pSB4K5] (4)White Colony
R0011-E0240(2) [pSB4K5] (1)Green Colony
R0011-E0240(2) [pSB4K5] (2)White Colony
R0011-E0240(2) [pSB4K5] (3)White Colony
R0011-E0240(2) [pSB4K5] (4)White Colony
I20260 [pSB4K5] (1)Green Colony
I20260 [pSB4K5] (2)Green Colony
I20260 [pSB4K5] (3)Green Colony
Sequence
NameConcentration
SΔTMD1-E0840(1) A28.9 ng/µL
SΔTMD1-E0840(1) B25.3
SΔTMD1-E0840(3) A26.6
SΔTMD1-E0840(3) 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-E0240(1) [pSB4K5] (1)
R0011-E0240(1) [pSB4K5] (2)
R0011-E0240(1) [pSB4K5] (3)
R0011-E0240(1) [pSB4K5] (4)
R0011-E0240(2) [pSB4K5] (1)
R0011-E0240(2) [pSB4K5] (2)
R0011-E0240(2) [pSB4K5] (3)
R0011-E0240(2) [pSB4K5] (4)
I20260 [pSB4K5] (1)
I20260 [pSB4K5] (2)
I20260 [pSB4K5] (3)
Restriction Digestion
NameSample2 bufferBSAEnzyme 1Enzyme 2MilliQTotal
R0011-E0240(1) [pSB4K5] (1) [EP]50 µL60.6EcoRI0.3PstI0.32.860
R0011-E0240(1) [pSB4K5] (2) [EP]5060.6EcoRI0.3PstI0.32.860
R0011-E0240(1) [pSB4K5] (3) [EP]5060.6EcoRI0.3PstI0.32.860
R0011-E0240(1) [pSB4K5] (4) [EP]5060.6EcoRI0.3PstI0.32.860
R0011-E0240(2) [pSB4K5] (1) [EP]5060.6EcoRI0.3PstI0.32.860
R0011-E0240(2) [pSB4K5] (2) [EP]5060.6EcoRI0.3PstI0.32.860
R0011-E0240(2) [pSB4K5] (3) [EP]5060.6EcoRI0.3PstI0.32.860
R0011-E0240(2) [pSB4K5] (4) [EP]5060.6EcoRI0.3PstI0.32.860
I20260 [pSB4K5] (1) [EP]5060.6EcoRI0.3PstI0.32.860
I20260 [pSB4K5] (2) [EP]5060.6EcoRI0.3PstI0.32.860
I20260 [pSB4K5] (3) [EP]5060.6EcoRI0.3PstI0.32.860
Electrophoresis
No.NameLengthResults
1I20260 [pSB4K5] (1) [EP]960, 4339
2I20260 [pSB4K5] (2) [EP]960, 4339
3I20260 [pSB4K5] (3) [EP]960, 4339
4R0011-E0240(1) [pSB4K5] (1) [EP]980 3378
5R0011-E0240(1) [pSB4K5] (2) [EP]980 3378
6R0011-E0240(1) [pSB4K5] (3) [EP]980 3378×
7R0011-E0240(1) [pSB4K5] (4) [EP]980 3378×
8R0011-E0240(2) [pSB4K5] (1) [EP]980 3378
9R0011-E0240(2) [pSB4K5] (2) [EP]980 3378×
10R0011-E0240(2) [pSB4K5] (3) [EP]980 3378×
11R0011-E0240(2) [pSB4K5] (4) [EP]980 3378×
12I20260 [pSB4K5] (1) [EP]960, 4339
13I20260 [pSB4K5] (2) [EP]960, 4339
KyotoExp100806-1.png

White colonies are not inserted R0011 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)
NameWaterMgSO4dNTPs10xBufferPrimer VF2Primer VRTemplate SSam7,ΔTMD1-E0840 failed (50ng/µL)KOD plus ver.2Total
SSam7,ΔTMD1-E0840 (1)323551.51.51150
SSam7,ΔTMD1-E0840 (2)323551.51.51150
94℃2min
98℃10s25 cycles
68℃4min
Add DpnI 2µL
Incubate1h
4℃forever
Transformation
NameWellSampleCompetent CellTotalPlateIncubationColony
SSam7,ΔTMD1-E0840 (1)-4 µL5054LB (Kan+)08/06-08/09
SSam7,ΔTMD1-E0840 (2)-45054
I202602-17-F25052
2-I-525052LB (Amp+)


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

Miniprep
Nameconcentration
I20260 [pSB4K5]116.2 ng/µL
R0011-E0240 [pSB4K5]146.6
Transfotrmation
SampleSampleCompetent CellTotalPlateIncuvationResults
I20260 [pSB4K5]2 µLKRX5052LB (Kan+)08/09 18:00-08/10 12:00
R0011-E0240 [pSB4K5]2KRX5052
Restriction Eigestion and Ethanol Precipitation

To use R0011 for next ligation, we digested it by EcoRI and PstI

NameSample10x BufferBSAEnzyme 1Enzyme 2MilliQTotalIncubation
R0011 [EP]5060.6EcoRI0.5PstI0.52.460At 37℃ 08/09 16:20-18:20

After restriction enzyme digestion, we did ethanol precipitation.

Ligation and Transformation
SampleCompetent cellTotalPlateIncuvationColony
R0011 [pSB4K5, KRX]2 µLKRX5052LB (Kan+)08/09 20:00-08/10 09:00
R0011 [pSB4K5</partrinfo>, C2]2C25052


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

Culture

Cultured I20260 [pSB4K5, R0011-E0240 [pSB4K5], R0011 [pSB4K5, KRX], and R0011 [pSB4K5</partrinfo>, C2].

Minprep
NameConcentration
SSam7,ΔTMD1-E0840 (1-1)9.9 ng/µL
SSam7,ΔTMD1-E0840 (1-2)27.3
SSam7,ΔTMD1-E0840 (2-1)43.2
SSam7,ΔTMD1-E0840 (2-2)34.7
Culture and Master Plate

At 37℃ 08/09 18:00-08/10 9:00


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

No.MediumCloudIncubation
1KanamycinAt 37℃, 08/10 20:00-08/11 9:00
Ampicillin×
2Kanamycin
Ampicillin
3Kanamycin
Ampicillin×
4Kanamycin
Ampicillin×
5Kanamycin
Ampicillin×
6Kanamycin
Ampicillin
7Kanamycin
Ampicillin×

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 R0011 [pSB4K5, C2], SRRz 1', 3'
NameConcentration
R0011 [pSB4K5, C2] (1)31.2 ng/µL
R0011 [pSB4K5, C2] (3)29.9
Restriction Digestion and electrophoresis of R0011 [pSB4K5, C2]
NameEcoRIPstI
10.2-
2-0.2
30.20.2
N--
No.NameLengthResults
1R0011 [pSB4K5, C2] (1-1)
2R0011 [pSB4K5, C2] (1-2)
3R0011 [pSB4K5, C2] (1-3)
4R0011 [pSB4K5, C2] (1-N)
5R0011 [pSB4K5, C2] (2-1)
6R0011 [pSB4K5, C2] (2-2)
7R0011 [pSB4K5, C2] (2-3)
8R0011 [pSB4K5, C2] (2-N)
KyotoExp100811-1.png

Each enzyme correctly cut samples.

Screening PCR of SRRz
No.NameResults
1None
2Control B0015
3Control J06702
4Control B0015
5-24SRRz-B0015×

Marker: Lambda Marker

KyotoExp100811-2.png
KyotoExp100811-3.png

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

Thursday, August 12 By: Wataru, Ken

Restriction Digestion and electrophoresis of B0015
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

Maker: Lambda, 100bp

KyotoExp100812-1.png

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


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

Miniprep of SSam7,ΔTMD1-E0840
NameConcentration
SSam7,ΔTMD1-E084029.6 ng/µL
Point mutation PCR of SSam7,ΔTMD1-E0840
NameTemplate10xbufferdNTPsMgSO4Primer Fwd.Primer Rev.MilliQKOD plus ver.2Total
SΔTMD1-E0840 (1)1.55531.51.531.5150
SΔTMD1-E0840 (2)1.55531.51.531.5150
Control1.55531.51.532.5-50
94℃2min
98℃10s30cycles
55℃30s
68℃3.5min
4℃forever
Restriction Digestion by DpnI from 17:50 to 18:50
Electrophoresis
Name
SΔTMD1-E0840 (1)
SΔTMD1-E0840 (2)
Control

Marker: Lambda, 100bp

KyotoExp100819-1.png
Ligation and Transformation
NameColony
SΔTMD1-E0840 (1)
SΔTMD1-E0840 (2)
Control×


Friday, August 20 By: Wataru, Ken

Making Culture and Master Plate of SΔTMD1-E0840
Miniprep
NameConcentration
B001541.1 ng/µL
PCR of SRRz
Name10xBufferMgS04dNTPTemplatePrimer Fwd.Primer Rev.MilliQKOD plus ver.2Total
SRRzSam7 (1)5 µL355F11.51.528150
SRRzSam7 (2)5355F21.51.528150
SRRzSam7 (3)5355F11.51.528150
SRRzSam7 (4)5355F21.51.528150
SRRzSam7 (5)5355F11.51.528150
SRRzSam7 (6)5355F21.51.528150
94℃2min
98℃10s30cycles
55℃30s
68℃2min
4℃forever
Electrophoresis
Name
SRRzSam7 (1)
SRRzSam7 (3)
SRRz Sam7(5)
SRRzSam7 (2)
SRRzSam7 (4)
SRRzSam7 (6)
KyotoExp100820-1.png

Discussion: Primer F1 might be better than F2, because the bands of 1, 2 and 3 were clearer. We decided to use sample 1 and 3. Their bands were clearer in the three.

PCR Purification
NameConcentration
SRRzSam7 (1)134.0 ng/µL
SRRzSam7 (3)69.0
Restriction Digestion
NameSample10xBuffer100xBufferEcoRIXbaISpeIMilliQTotalIncubation
B0015 [EX]50 µL60.60.40.4-2.66017:45-18:45
SRRzSam7 (1) [EP]5060.60.4-0.42.660
SRRzSam7 (3) [EP]5060.60.4-0.42.660
Purification
NameConcentration
SRRzSam7 (1) [EP]109.0 ng/µL
SRRzSam7 (2) [EP]110.0
B001525.5
Ligation and Transformation

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

Miniprep
Sample numberConcentration
SΔTMD1-E0840 (1-1)58.9 ng/µL
SΔTMD1-E0840 (2-2)49.9
Sequence

Sample: SΔTMD1-E0840 (1-1). SΔTMD1-E0840 (2-2), I20260 [pSB4K5] Discussion: The sequencing was in success and the results were desirable. It meant the point mutation was succeeded and sequence of I20260 [pSB4K5] was confirmed. We decided to use SΔTMD1-E0840.

Screening PCR of SRRzSam7-B0015
90℃10min
94℃30s35cycles
50℃30s
72℃1.5min
72℃4min
4℃hold
No.Name
1-13SRRzSam7-B0015
CControl: B0015
NNone

Marker: Lambda, 100bp

KyotoExp100823-1.png

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

Deletion PCR of SΔTMD1-E0840 (2-2)
NameSample10xBufferdNTPsPrimer Fwd.Primer Rev.TemplateWaterKOD-plus-Total
rrSΔTMD1-E0840 (1)5 µL51.51.5135150
rrSΔTMD1-E0840 (2)551.51.5135150
rrSΔTMD1-E0840 (Control)551.51.5135-50
94℃2min
94℃10s35cycles
56℃30s
68℃3.5min
4℃forever
Restriction Digestion (DpnI)
SampleDpnITotalIncubation
25 µL12619:00-20:10
Ligation
NameSampleWaterLigation highT4 KinaseTotalIncubation
rrSΔTMD1-E0840 (1)3 µL6511520:15-21:15
rrSΔTMD1-E0840 (2)36511520:15-21:15
rrSΔTMD1-E0840 (Control)365115
Transformation

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

Retry of deletion PCR of SΔTMD1-E0840
NameSample10xBufferdNTPsMgSO4Primer1Primer2TemplateWaterKOD-plus-Total
rrSΔTMD1-E0840 (1)5 µL531.51.5132150
rrSΔTMD1-E0840 (2)5531.51.5132150
Control5531.51.5132150
94℃2min
94℃10s35cycles
58℃30s
68℃3.5min
4℃hold
Restriction Digestion (DpnI)

14:15-15:15

Electrophoreis
LaneName
1rrSΔTMD1-E0840 (1)
2rrSΔTMD1-E0840 (3)
CrrSΔTMD1-E0840 (Control)

Marker: 100bp, Lambda.

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 Restriction Digestion were completed successfully.

Ligation
Point mutation of SRRz
Name10xdNTPsMgSO4Primer1Primer2TemplateWaterKOD-plus-total
SRRzSam7-B0015 (1)5531.51.5132150
SRRzSam7-B0015 (2)5531.51.5132150
SRRzSam7-B0015 (Control)5531.51.5132150
94℃2min
98℃10s30cycles
55℃30s
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

Sample10xBufferdNTPsMgSO4VF2VRTemplateWaterKOD-plus-Total
E0240 (1)5531.51.5131.5150
E0240 (2)5531.51.5131.5150
PCR Purification
NameConcentration
E0240 (1)5.5 x 50 ng/µL
E0240 (2)5.2 x 50
Restriction Digestion (EcoRI, PstI) and Gel Extraction
NameConcentration
E0240 (1) 28.8 ng/µL
E0240 (2) 26.4
Transformation

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

Making culture and Master plate
NameColony
rrSΔTMD1-E0840 (1)
rrSΔTMD1-E0840 (2)
rrSΔTMD1-E0840 (Control)×
SRRzSam7-B0015 (1)
SRRzSam7-B0015 (2)
SRRzSam7-B0015 (Control)×
Miniprep
NameConcentration
pSB4K529.0 ng/µL
Restriction Digestion
Sample nameTemplate10xbuffer100xbufferEcoRISpeIPstIWaterTotal
pSB4K55060.60.40.4-2.660
R0011 [pSB4K5]1040.4-0.30.32540
Purification
Sample NameConcentration
pSB4K518.4 ng/µL
R0011 [pSB4K5]8.6
Ligation of E0240 and pSB4K5, Transformation

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

Miniprep
Sample nameConcentration
J23116 (RPU0.7)44.5 ng/µL
Restriction Digestion
NameTemplate10xbuffer100xbufferSpeIPstIWaterTotal
J23116 (RPU0.7)2540.40.30.31040
Purification of
NameConcentration
J23116 (RPU0.7)49.8 ng/µL


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

Making master plate of E0240 [pSB4K5]
Sample NameConcentration
rrSΔTMD1-E0840 (1-2)20.9 ng/µL
SRRz-B0015 (1-1)16.4
Restriction Digestion
NameTemplate10xbuffer100xbufferXbaIPstIWaterTotalIncubation
rrSΔTMD1-E0840 (1-2)45 µL60.60.30.37.86013:20-14:20
SRRz-B0015 (1-1)4560.60.30.37.860
Purification
rrSΔTMD1-E0840 (1-2)44.7 ng/µL
SRRz-B0015 (1-1)56.1
Ligation and Transformation
Name
R0011-rrSΔTMD1-E0840 (1-2)
J23116 (RPU0.7)- rrSΔTMD1-E0840 (1-2)
R0011-SRRz-B0015 (1-1) [pSB4K5]

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

Making culture and Master plate
NameColony R0011-rrSΔTMD1-E0840Many colonies
R0011-rrSΔTMD1-E0840 (Control)Some colonies
J23116 (RPU0.7)- rrSΔTMD1-E0840Many colonies
J23116 (RPU0.7)- rrSΔTMD1-E0840 (Control)Many colonies
R0011-SRRz-B0015 (1-1) [pSB4K5]No colony
R0011-SRRz-B0015 (1-1) [pSB4K5] (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
NameConcentration
J23105 (RPU0.3)48.5 ng/µL
R0011-rrSΔTMD1-E0840107.3
Restriction Digestion
Gel Extraction of R0011-rrSΔTMD1-E0840 (Electrophoresis for 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 J23105 (RPU0.3) and R0011-rrSΔTMD1-E0840
NameConcentration J23105 (RPU0.3)5.8 ng/µL
R0011-rrSΔTMD1-E08407.8
Ligation and Transformation
InsertVector
R0011-rrSΔTMD1-E0840J23105 (RPU0.3)


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

Making culture and Master plate
NameColony
R0011-rrSΔTMD1-E0840-J23105 (RPU0.3)Many colonies
R0011-rrSΔTMD1-E0840-J23105 (RPU0.3) (Control)Many colonies
Screenig PCR of R0011-rrSΔTMD1-E0840-J23105 (RPU0.3)
  • Sample: 1-13
  • Control: Positive (B0015)
  • Maker: lambda, 100

Discussion: All of the sample except sample 10 might be self-ligation products of J23105 (RPU0.3).

Miniprep
SRRz-B0015 (1-1)33.8 ng/µL
pSB4K556.0
Restriction Digestion of SRRz and pSB4K5
NameTemplate10xbuffer100xbufferEcoRIPstIWaterTotalIncubation
SRRz-B0015 (1-1)20 µL40.40.30.3154013:25-14:30
pSB4K52040.40.30.31540
Purification
SRRz-B0015 (1-1)6.5 ng/µL
pSB4K516.8

Ligation and transformation

  • Insert: SRRz-B0015 (1-1)
  • Vector: pSB4K5

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

Making culture and Master plate
SRRz-B0015 (1-1) [pSB4K5]13 colonies
SRRz-B0015 (1-1) [pSB4K5] (Control)13 colonies
Screening PCR of rSRRz low

Sample: (1-13) SRRz-B0015 (1-1) [pSB4K5] Maker: Lambda, 100 Control: Positive (B0015), Neganive Discussion: From sample 1, two vectors might be ligated. Sample 3 and 4, SRRz-B0015 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
  • R0011-rrSΔTMD1-E0840 (1)
  • R0011-rrSΔTMD1-E0840 (3)
  • rrSΔTMD1-E0840 (1-1)
  • rrSΔTMD1-E0840 (1-2)
  • SRRz-B0015 (1-1) [pSB4K5]
  • SRRz-B0015 (1-2) [pSB4K5]
  • R0011-E0240 [pSB4K5]