Team:Kyoto/Notebook

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Notebook

Tuesday, July 20

By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto

1. Solubilization of antibiotics.

for Ampicillin(Amp): add 1.0g Amp to 20ml MilliQ (final concentration is 50mg/ml).
for Kanamycin(kan): add 0.5g Kan to 10ml MilliQ (final concentration is 50mg/ml).
Dispense 1.1ml of the solution into 1.5ml tubes.
Store in the freezer (-20℃).

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

3. Transformation of iGEM Parts.

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 (Kanamycin+)		×

"1-18-C" means well 18C in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
Discussion
A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Ampicillin+).
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 Yamamoto

1. Culture plates in which colonies was observed at 37℃ from 07/21 20:50 to 07/22 17:00.

2. Make a master plate of the above plates.

3. Retry Transformation of iGEM Parts.

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

4. PCR for S-R-Rz/Rz1 and S

Dilute λDNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template λDNA was 5ng/µl.

No.	Water	25mM MgSO4	2mM dNTPs	10xBuffer for KOD Plus ver.2	TemplateDNA (5ng/µl)	Primer S-R-Rz/Rz1 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

Forward Primer of S-R-Rz/Rz1 and S is common.
PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.

Thursday, July 22

By: Wataru

1. Electrophoresis of the PCR products for 40min.

File:KyotoEXP100722-1.png

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

2. Miniprep of iGEM Parts.

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

Discussion
The concentration of all samples was very week. Probably our shaking incubation was week.

3. Culture plates and make master plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo> from 7/22 17:00 to 7/23 10:00.

Friday, July 23

By: Wataru, Tomo, Makoto

1. Miniprep of iGEM Parts.

Name	Concentration(ng/µl)
<partinfo>pSB4K5</partinfo>	79.2
<partinfo>B0015</partinfo>	-

Discussion
We lost <partinfo>B0015</partinfo> by our mistake.
The concentration of <partinfo>pSB4K5</partinfo> is high, so this condition of shaking incubation is moderate.

2. Picked up 1, 3, 5, 7 of the products of PCR, and purified by PCR-purification.

Sample	Concentration (ng/µl)	New Name
1	18.6	-
3	77.6	S₁
5	33.6	-
7	65.4	S₂

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

3. Retry of PCR of S-R-Rz/Rz1.

Sample	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℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.

4. Digested <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, PstI to check function of our Restriction enzymes.

Sample	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

|-

5. Electrophoresis of above sample for 35min.

Discussion
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.

6. To insert S gene to GFP, we digested the PCR products of S gene by EcoRi and SpeI, and GFP by EcoRl and XbaI.

Sample	10×Buffer	Enzyme 1	Enzyme 2	MilliQ	Total	Incubation
S₁	11µl	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>(GFP)	45	5	EcoRI 0.2	XbaI 0.2	0	50

After PCR purification, evaporated them and diluted 3ul.

7. Ligated over night.

Sample	Vector	Insert	Ligation High	Total
S-GFP₁	<partinfo>E0840</partinfo> 0.5µl	S₁ 0.5	1	2
S-GFP₂	<partinfo>E0840</partinfo> 0.5	S₂ 0.5	1	2

Monday, July 26

By: Wataru, Tomonori, Makoto

1. Electrophoresis of PCR products

Discussion
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.

2. PCR purification

Sample	Concentration (ng/µl)	New Name
4	51.6
5	59.3
6	59.6

3. Transformation of iGEM Parts

Well	Sample (µl)	Competent Cell (µl)	Total (µl)	Plate	Incubation	Result
1-12-M	1	20	21	LB (Amplicillin+)	At 37℃ 7/26 - 7/27	×
2-17-F	1	20	21	LB (Kanamycin+)		×
1-5-E	1	20	21	LB (Kanamycin+)		×