Team:Kyoto/Notebook

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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 MilliQ	Mix 0.5g Kan and 10ml MilliQ	Final 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

Name	Well	Sample (µl)	Competent Cells (µl)	Total (µl)	Plate	Incubation	Result
<partinfo>J23100</partinfo>	1-18-C	1	20	21	LB (Amp+)	At 37℃, 7/20 20:50 - 7/21 17:00	○
<partinfo>J23105</partinfo>	1-18-M	1	20	21			○
<partinfo>J23116</partinfo>	1-20-M	1	20	21			○
<partinfo>R0011</partinfo>	1-6-G	1	20	21			○
<partinfo>E0840</partinfo>	1-12-O	1	20	21			○
<partinfo>J06702</partinfo>	2-8-E	1	20	21			○
<partinfo>pSB4K5</partinfo>	1-5-G	1	20	21			×
<partinfo>B0015</partinfo>	1-23-L	1	20	21	LB (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

Name	Well	Sample (µl)	Competent Cells (µl)	Total (µl)	Plate	Incubation	Result
<partinfo>pSB4K5</partinfo>	1-5-G	1	20	21	LB (Kan+)	At 37℃ 7/21 20:50 - 7/22 16:30	○
<partinfo>B0015</partinfo>	1-23-L	1	20	21	LB (Kan+)	At 37℃ 7/21 20:50 - 7/22 16:30	○

PCR for S-R-Rz/Rz1 and S

No.	Water	25mM MgSO4	2mM dNTPs	10xBuffer for KOD Plus ver.2	TemplateDNA (5ng/µl)	Primer Forward (10µM)	Primer S-R-Rz/Rz1 Reverse (10µM)	Primer S Reverse (10µM)	KOD Plus ver.2	Total
1	28µl	3µl	5µl	5µl	5µl	1.5µl	1.5µl	-	1µl	50µl
2	28µl	3µl	5µl	5µl	5µl	1.5µl	1.5µl	-	1µl	50µl
3	28µl	3µl	5µl	5µl	5µl	1.5µl	-	1.5µl	1µl	50µl
4	28µl	3µl	5µl	5µl	5µl	1.5µl	-	1.5µl	1µl	50µl
5	28µl	3µl	5µl	5µl	5µl	1.5µl	1.5µl	-	1µl	50µl
6	28µl	3µl	5µl	5µl	5µl	1.5µl	1.5µl	-	1µl	50µl
7	28µl	3µl	5µl	5µl	5µl	1.5µl	-	1.5µl	1µl	50µl
8	28µl	3µl	5µl	5µl	5µl	1.5µl	-	1.5µl	1µl	50µl

PCR condition
94℃	2min
98℃	10sec	30 cycles
55℃	30sec
68℃	4min
4℃	forever

Thursday, July 22 By: Wataru

Electrophoresis of the PCR products for 40min

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

Miniprep

Name	Concentration(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

Name	Concentration(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.	Name	Concentration (ng/µl)	New Name
1	S-R-Rz/Rz1	18.6	-
3	S	77.6	S₁
5	S-R-Rz/Rz1	33.6	-
7	S	65.4	S₂

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.	Water	25mmol/l MgSO4	2mmol/l dNTPs	10×Buffer for KOD plus ver.2	Template DNA (5ng/µl)	Primer S-R-Rz/Rz1 Forward (10µmol/l)	Primer S-R-Rz/Rz1 Reverse (10µmol/l)	KOD plus ver.2	Total
1	28µl	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

PCR condition
94℃	2min
98℃	10sec	30 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.	10xBuffer	BSA	Enzyme	MilliQ	Total	Incubation
1	5µl	1	EcoRI 0.1	3.6	10	At 37℃ 7/23 18:00 - 7/23 18:30
2	5	1	XbaI 0.1	3.6	10
3	5	1	SpeI 0.1	3.6	10
4	5	1	PstI 0.1	3.6	10
5	5	1	-	3.7	10

Electrophoresis of above sample for 35min

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>

Name	Sample Volume (µl)	10×Buffer	Enzyme 1	Enzyme 2	MilliQ	Total	Incubation
S₁	11	5	EcoRI 0.2	SpeI 0.2	33.6	50	At 37℃ for 2h
S₂	11	5	EcoRI 0.2	SpeI 0.2	33.6	50
<partinfo>E0840</partinfo>	45	5	EcoRI 0.2	XbaI 0.2	0	50

After PCR purification, evaporated them and diluted 3ul.

Ligation

Name	Vector	Insert	Ligation High	Total
S-E0840₁	<partinfo>E0840</partinfo> 0.5µl	S₁ 0.5	1	2
S-E0840₂	<partinfo>E0840</partinfo> 0.5	S₂ 0.5	1	2

Team:Kyoto/Notebook

From 2010.igem.org

Contents

Index

Notebook

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

Solubilization of Antibiotics

Making plates for LB (Amp+) and LB (Kan+)

Transformation

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

PCR for S-R-Rz/Rz1 and S

Thursday, July 22 By: Wataru

Electrophoresis of the PCR products for 40min

Miniprep

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

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

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

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

Electrophoresis of above sample for 35min

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>

Ligation