Team:Kyoto/Notebook

From 2010.igem.org

Revision as of 13:05, 14 September 2010 by Naoi (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

LastModified: 2010-9-14

Index

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+)		×

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

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

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>	-

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

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.

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

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+)		×

4. Culture of 1-6-G, 1-12-O, and 1-23-L

Monday, July 26

By: Wataru, Tomonari, Makoto

Electrophoresis of PCR products

3①3②4.5①4.5②6①6②

Discussion

At the condition 4.5②　and 6②、S-R-Rz is amplified very much. So we decided to use them inexperience.

PCR purification

Sample number	Concentration(ng/µL)
4.5②	51.6
6①	59.3
6②	59.6

Transformation

Sample	Conc(/µL)	Sample Volume(µL)	Competent Cell(µL)	Total	Plate	Incubation
1-12-M	-	1	20	21	LB amp	7/26~7/27
2-17-F	-	1	20	21	LB kan
1-5-E	-	1	20	21	LB kan

Culture

1-6-G, 1-12-O, 1-23-L

Tuesday, July 27

By: Wataru, Tomo, Kazuya, Ken, Naoi

Result of transformation

1-12-M	No colony
1-5-E
2-17-F

Colony of PCR of S-E840

To check that S GFP is correctly inserted, we did colony PCR.

Gel: Agarose Time: 35min Voltage: 100V Maker: 1K 100 1～6 S-GFP① 7～13 S-GFP② Posi E840（about 900bp） Nega None

1K 1 2 3 4 5 6 7 8 9 10 11 12 13 posi nega 100 File:KyotoExp100727.png

As a result, 1,3,5,6,11,12,13 are inserted S gene correctly. So, we decided to use 6 as S-E840①　and 11 as S-E840②.

Miniprep

Sample number	Concentration(ng/µL)
1-6-G	26.9
1-23-L	120.0
1-12-O	120.1

RE

	Sample volume	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
1-23-L	30	5	0.5	EcoRI	0.4	XbaI	0.3	13.7	50
4.5②	40	5	0.5		0.4		0.4	3.8	50
6②	40	5	0.5		0.4		0.4	3.8	50

Incubate 37℃ 16:45~18:00

Ligation

Transformation

Wednesday, July 28

1. Result of Transformation

SRRz①-DT	Many colonies
SRRz②-DT

2. Deletion PCR

To delete functional domain of S gene, we did deletion PCR.

Miniprep

Sample number	Concentration(ng/µ)
S-E840①	95.5
S-E840②	98.6

Diluted S-GFP①　and S-GFP②　20 times with water, and used as template DNA.

Deletion PCR

To delete functional region of S gene, we did deletion PCR.

	Water	25mM MgSO4	2mM dNTPs	10x buffer for KOD Plus ver.2	Primer Deletion F(10µM)	Primer Deletion R(10µM)	Template S-E840①	Template S-E840②	KOD Plus ver.2	Total
Δ1-1	28	3	5	5	1.5	1.5	5	-	1	50
Δ1-2	28	3	5	5	1.5	1.5	5	-	1	50
Δ2-1	28	3	5	5	1.5	1.5	-	5	1	50

PCR program

94℃	2min
98℃	10sec	35 cycles
55℃	30sec
68℃	4min
4℃	forever

3. RE

To check function of our Restriction enzymes, we digested S-E840① and S-E-840② by DpnI.

	Sample	fast digestion buffer	DpnI	MilliQ	Total
S-E840①	3	1	0.1	5.8	10
S-E840②	3	1	0.1	5.8	10

Electrophoresis

Gel: Agarose Time: 35min Voltage: 100V Maker: 1K 100

1 1k marker 2 not digested S-E840① 3 not digested S-E840② 4 digested S-E840① 5 digested S-E840② 6 100bp marker

1k 1 2 3 4 100

File:KyotoExp100728.png

Discussion

DpnI works correctly

Thursday, July 29

RE

	Sample volume	Fastdigestion buffer	Enzyme 1	MilliQ	Total
Δ1-1	50	6	DpnI 0.2	3.8	60
Δ2-1	50	6	DpnI 0.2	3.8	60

Incubate 7/29 9:40~7/29 11:00

Ligation and Pospholylation

	Sample	MilliQ	Ligation High	T4 Kinase	Total
Δ1-1	2	7	5	1	15
Δ2-1	2	7	5	1	15

Incubate 7/29 11:30~7/29 13:00

Transformation

Sample	Conc(/µL)	Sample Volume(µL)	Competent Cell(µL)	Total	Plate	Incubation
Δ1-1	-	3	30	33	LB amp	7/29~7/30
Δ1-1	-	3	30	33	LB amp	7/29~7/30

Friday, July 30

Result of transformation of Δ1 and Δ2

Many colonies are observed.

Team:Kyoto/Notebook

From 2010.igem.org

Contents

Index

Notebook

Tuesday, July 20

1. Solubilization of antibiotics.

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

3. Transformation of iGEM Parts.

Wednesday, July 21

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.

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

Thursday, July 22

1. Electrophoresis of the PCR products for 40min.

2. Miniprep of iGEM Parts.

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

1. Miniprep of iGEM Parts.

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

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

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

5. Electrophoresis of above sample for 35min.

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.

7. Ligated over night.

Monday, July 26

1. Electrophoresis of PCR products

2. PCR purification

3. Transformation of iGEM Parts

4. Culture of 1-6-G, 1-12-O, and 1-23-L

Monday, July 26

Electrophoresis of PCR products

PCR purification

Transformation

Culture

Tuesday, July 27

Colony of PCR of S-E840

Miniprep

RE

Ligation

Transformation

Wednesday, July 28

1. Result of Transformation

2. Deletion PCR

Miniprep

Deletion PCR

3. RE

Electrophoresis

Thursday, July 29

RE

Ligation and Pospholylation

Transformation

Friday, July 30