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Revision as of 16:31, 9 September 2012

Secretion Notebook

February 7

====Preculture==== by_???
We started preculture at 12:10.

February 8

March 1

March 2

March 3

PCR

	template	buffer	dNTPs	MgSO4	VF	VR	KOD plus	MilliQ	total
1	1	5	5	3	1.5	1.5	1	32	50
2	2	5	5	3	1.5	1.5	1	31	50

94℃, 2min
98℃, 10sec
50℃, 30sec
68℃, 1min
→30cycles

Miniprep

March 4

Sequence of tatABCD

Quick Taq	primer-f	promer-r sequence	template	MilliQ	total
25	1	1	1	23	50

Quick Taq	primer-f sequence	primer-r	template	MilliQ	total
25	1	1	1	23	50

Colony PCR of TMAO

buffer	dNTPs	NgSO4	primer-f	primerr-r	KOD plus	MilliQ	total
5	5	4	1.5	1.5	1	32	50

→ethanol precipitation

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 2.5min
→25cycles

Electrophoresis

1. 1kb ladder
2. tatABCD1
3. tatABCD2
4. TMAO
5. 1kb ladder

Restriction

TMAO	EcoR1	BufferH	BSA	MilliQ	total
10	0.2	3	0.3	16.5	30

TMAO	Xba1	Pst1	BudderM	BSA	MilliQ	total
10	0.2	0.2	3	0.3	16.3	30

at 37℃ for 1 hour

====Electrophoresis====

Transformation

pSB1C3	competent cell(made at 2/8)	total
5	100	105

March 5

Restriction

pSB1C3(Xba1, Spe1)	Pst1	BufferH	BSA	MilliQ	total
10	0.2	2	0.2	7.6	20
pSB1C3(Xba1, Spe1)	EcoR1	BufferH	BSA	MilliQ	total
10	0.2	2	0.2	7.6	20

at 37℃ for 1 hour
→Then we did ethanol precipitation

Ligation

Kil(EcoR1, Spe1)	pSB1C3(EcoR1)	Ligation High	total
5	1	3	9

at 16℃ for 1 hour

Transformation

Kil	competent cell	total
1	10	11

We used commercially available competent cells in this time.

PCR

TMAO

buffer	dNTPs	MgSO4	Primer-f	Primer-r	Template	KOD plus	MilliQ	total
5	5	3	1.5	1.5	1	1	32	50

Electrophoresis

(31)

Restriction

	Lacp	pSB3C5	EcoR1	Pst1	BufferH	BSA	MilliQ	total
1	20	0	0.5	0.5	3	0.5	5.5	30
2	0	20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1 hour
1→Ethanol precipitation　45.4µg/mL
2→Gel extraction　38.7µg/mL

Ligation

LacP	pSB3C5	Ligation High	total
10	2	6	18

at 4℃ for overnight

Transformation

Lacp+pSB3C5	competent cell	total
1	10	11

on ice for 30 mins.
heat shock at 42℃ for 60secs
on ice for 2 mins.
After we incubate with 200µL of SOC culture for 1 hour, we did plating on LB culture with CP

Restriction

GFP Plasmid	EcoR1	Spe1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

at 37℃ for 2 hours

Electrophoresis

1. Ladder 2µL
2. GFP Plasmid (already restricted) 2µL + Loading Dye 2µL + MilliQ 8µL
3. Ladder 2µL

PCR

torA signal and pspA pspAはコロニーPCR

Buffer	dNTPs	MgSO4	Primer-f	Primer-r	template(TMAO)	KOD plus	MilliQ	total
2.5	2.5	1.5	0.75	0.75	0.5	0.5	16	25

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec

electrophoresis

1. 100bp Ladder
2. torA signal (272bp)
3. pspA (969bp)
4. 100bp Ladder

March 6

Restriction

GFP	EcoR1	Spe1	BufferM	BSA	MilliQ	total
12	0.5	0.5	3	0.5	13.5	30

We did incubate at 37℃ for 1.5hours.
And we did gel extraction on 3/7 and get 40.0μg/mL GFP.

PCR

buffer	dNTPs	MgSO4	Primer-f	Primer-r	template	KOD plus neo	MilliQ	total
5	5	3	1.5	1.5	0.5	1	32.5	50

94℃　2min
98℃　10sec
60℃　30sec
68℃　(torA 10sec / pspA 30sec) 25 cycles
We used product of PCR on 3/5 of torA and pspA as template.

Restriction

Kil	EcoR1	Spe1	BufferM	BSA	MilliQ	total
10	0.3	0.3	3	0.3	16.1	30

DT	EcoR1	Xba1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

at 37℃ for 2 hours
→ purification 37.7ng/μL

Ligation

Kil	pSB1C3	Ligation High Ver.2	total
4	2	3	9

Kil : 350fmol
pSB1C3 : 29fmol

at 16℃ for overnight

March 7

====Electrophoresis====

1. 1kb ladder 2µL
2. pspA (PCR product)2.5µL + Loading Dye 0.5µL
3. GFP 30µL + Loading Dye 6µL
4. 1kb ladder 2µL

The GFP was gel extracted on 3/6 and concentrated by Vacuum in 150µg/mL

Ligation

VectorDNA	GFP	Ligation High Ver.2	total
5	15	10	30

Restriction

torA	EcoR1	Spe1	bufferM	BSA	MilliQ	total
10	0.3	0.3	3	0.3	16.1	30

pspA	EcoR1	Spe1	bufferM	BSA	MilliQ	total
5	0.3	0.3	3	0.3	21.1	30

at 37℃ for 1.5 hours

Purification

torA→31.8ng/µL
pspA→49.3ng/µL

Ligation

torA	pSB1C3	Ligation High Ver.2	total
3	3	3	9

pspA	pSB1C3	Ligation High Ver.2	total
4	2	3	9

at 4℃, for overnight

torA→31.8ng/µL×3µL=95.4ng=0.529pmol
pSB1C3→19.4ng/µL×3µL=58.2ng=0.042pmol
pspA→49.3ng/µL×4µL=197.2ng=0.308pmol
pSB1C3→19.4ng/µL×2µL=38.8ng=0.029pmol

March 8

Restriction

pSB4K5	EcoR1	Spe1	BufferM	BSA	MilliQ	total
20	0.2	0.2	3	0.2	6.4	30

at 37℃ for 1 hour.
→Purification : 36.6ng/µL

Ligation

Kil	pSB4K5	Ligation High Ver.2	total
10	1	5	16

at 4℃ for overnight

Kil→37.7ng/µL×10µL=377ng=879fmol
pSB4K5→36.6ng/µL×1µL=36.6ng=86fmol

Liquid culture

Lacp + pSB3C5 -1, 2

Transformation

torA	pspA	competent cell	total
1	0	10	11
0	1	10	11

We use commercially available competent cells in this time.

March 9

Restriction

tatABCD	Xba1	Pst1	BufferM	BSA	MilliQ	total
10	0.2	0.2	3	0.3	16.3	30

at 37℃ for 1hour
→purification : 75.0μg/mL (1.11 260/280 , 0.81 260/230)

Miniprep

lacP + pSB3C5 1 80.5μg/mL (1.78 260/280 , 2.00 260/230)
lacP + pSB3C5 2 107.2μg/mL (1.83 260/280 , 1.90 260/230)

Colony PCR

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

94℃ 2min
94℃ 30sec
55℃ 30sec
68℃ 6sec
25cycles

Ligation

tatABCD	constP J23107	Ligation High Ver.2	total
5	1	3	9

tatABCD : 227fmol
constP J23107 : 21fmol

Transformation

	pspA	torA	Kil	competent cell
1	1	0	0	10
2	0	1	0	10
3	0	0	1	10

Miniprep

4mL of plusgrow which had been cultured for overnight.
pSB4K5 : 80.5μg/mL

March 10

Screening PCR

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

Then we did electrophoresis to confirm.
1.1kb ladder
2.kil (649bp)
3,4,5, pspA (969bp)
6,1kb ladder

1, 100bp ladder
2,3,4, torA signal

Restriction

LacP-pSB3C5	Spe1	Pst1	BufferM	BSA	MilliQ	total
10	0.2	0.2	2	0.2	7.4	20

for 2.5 hours at 37℃
→purification 29.0ng/μL

torA	Xba1	Pst1	BufferM	BSA	MilliQ	total
10	0.2	0.2	2	0.2	7.4	20

for 2.5 hours at 37℃
→purification 91.8ng/μL

Ligation

torA	Lacp-pSB3C5	Ligation High Ver.2	total
4	2	3	9

torA : 929fmol
Lacp-pSB3C5 : 284fmol
for overnight at 4℃

March 11

Miniprep

We used 3μL of plus grow that we had cultured for overnight.
torA : 62.8ng/μL

Screening PCR

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

Electrophoresis

1. 1kb ladder
2〜11. pspA (969bp)
12. 1kb ladder

The results were shown as photograph in the right.

It seemed that there were shorter sample than expected sample, so we did electrophoresis with pspA which was product of PCR and pspA which had already cut with EcoR1 and Spe1.

1.1kb ladder
2. pspA (PCR product)
3, pspA (Eco, Spe)
4〜6, pspA (colony PCR)
7,1kb ladder

The results were shown as photograph in the right.

March 12

Transformation

DT(1ng/μL)	DT(0.1ng/μL)	Kil	lacP-torA	MilliQ	competent cell	total
1	0	0	0	0	20	21
0	1	0	0	0	20	21
0	0	5	0	0	50	51
0	0	0	5	0	50	51
0	0	0	0	1	20	21

Restriction

pspA	EcoR1	Spe1	BufferM	BSA	MilliQ	total
10	0.5	0.5	3	0.3	15.7	30

at 37℃ for 4 hours
→ We did purification and got 48.3ng/μL pspA.

Ligation

pspA	pSB1C3	MilliQ	Ligation High Ver.2	total
4	2	0	3	9
2	2	0	2	6
0	2	2	2	6

March 13

Miniprep

pSB1C3 74.2µg/mL 1.65 (260/280) 1.31 (260/230)

March 14

Restriction

pSB1C3	EcoR1	Spe1	BufferM	BSA	MilliQ	total
20	0.2	0.2	4	0.4	15.2	40

We did gel extraction and got 47.2ng/µL pSB1C3 but we did not cut out RFP.

Ligation

torA	pSB1C3	Ligation High Ver.2	total
4	2	3	9
MilliQ	pSB1C3	Ligation High Ver.2	total
4	2	3	9

at 16℃, for 1 hour

torA : 0.707pmol
pSB1C3 : 0.068pmol

Liquid culture

We cultured Lacp-pSB3C5 1,2,3,4,5 (CP tolerance)on culture with Amp.
→Only 4 which did not be cultured succeeded.

March 15

Liquid culture

We cultured Lacp-pSB3C5 6,7,8,9,10 on culture with ampicillin.
→6,8,9,10 were succeeded.

Restriction

GFP	EcoR1	Spe1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

DT	EcoR1	Xba1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

for 2 hours at 37℃.

Miniprep

pspA (pSB1C3) 40.5ng/µL

Ligation

pspA	DT	Ligation High Ver.2	total
5	1.5	3	9.5

pspA : 385fmol
DT : 36fmol

Transformation

J23107-tatABCD	DT (0.1ng/µL)	DT (0.01ng/µL)	pspA-DT	competent cells on 3/15	total
2	0	0	0	20	22
0	2	0	0	20	22
0	0	2	0	20	22
0	0	0	2	20	22

Screening PCR

Kil, pspA and torA

Quick Taq	Primer-r	Primer-f	MilliQ	total
25	1	1	23	50

March 16

Miniprep

torA (pSB1C3) 68.8ng/µL
Kil (pSB4K5) 92.7ng/µL
torA was red for some reason. We do not know why.

Colony PCR

Quick Taq	Primer-r	Primer-f	MilliQ	total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 6sec
→25cycles

Electrophoresis

The results were shown as photograph in the right.

Checking Transformation Efficiency

competent cells that were made on March 15.
DNA : 0.02ng → 668 colonies Transformation Efficiency : 3.3×10^7
DNA : 0.2ng → 1739 colonies Transformation Efficiency : 8.7×10^6

Restriction

pSB1C3	EcoR1	Spe1	BSA	BufferM	BufferH	MilliQ	total
20	0.2	0.2	0.3	3	0	6.3	30
5	0.2	0	0.2	0	2	12.6	20

at 37℃, for 2 hours.
We did gel extraction for product with EcoR1, Spe1. We got 46.1ng/µL pSB1C3.

Kil(pSB4K5)	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

for overnight at 37℃.
We did this to confirm.

pspA (pSB1C3)	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

for overnight at 37℃.
We did this to confirm.

Ligation

torA	pSB1C3	Ligation High Ver.2	total
4	2	3	9

MilliQ	pSB1C3	Ligation High Ver.2	total
4	2	3	9

at 16℃, for overnight

torA→767fmol
pSB1C3→68fmol

March 17

Miniprep

J23107-tatABCD 72.7ng/µL
pspA-DT 50.5ng/µL

Checking the Insert

J21037-tatABCD	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

Success.

pspA-DT	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

Failed.

March 19

Restriction

DT	EcoR1	Xba1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30

We did Gel extraction and got 17.2ng/µL of DT.

Ligation

Kil	DT	Ligation High Ver.2	total
10	2	6	18

We did this for an hour at 16℃.

Restriction

GFP	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.5	0.5	0.5	3	15.5	30

We did this for 4 hours at 37℃

Ligation

pspA	DT	Ligation High Ver.2	total
5	5	5	15

pspA	pSB1C3	Ligation High Ver.2	total
4	1	3	8

We did these for an hour at 16℃.

pspA (5µL)→377fmol
DT→39fmol
pspA (4µL)→339fmol
pSB1C3→34fmol

Transformation

pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT

March 20

Screaning PCR

Quick Taq	Primer-R	Primer-F	MilliQ	total
25	1	1	23	50

pspA→○
pspA-DT→○
GFP-DT→○
torA→×
Kil-DT 6 of 8 sumples→○

Quick Taq	VR	VF	MilliQ	total
25	1	1	23	50

pspA→○
pspA-DT→×

Restriction

torA	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.3	0.3	0.3	3	16.1	30

DT	EcoR1	Xba1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30

We did this for overnight at 37℃. And we did purification.
torA 34.2ng/µL
DT 28.0ng/µL

March 21

Miniprep

GFP-DT-1 : 77.7ng/µL
GFP-DT-2 : 67.6ng/µL

Restriction

pSB1C3	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30
5	0.2	0	0.2	2	12.6	20

We did this for 3 hours at 37℃, and then we did gel extraction. We got 25.1ng/µL pSB1C3

GFP-DT	EcoR1	Pst1	Xba1	BSA	BufferM	MilliQ	total
5	0.2	0.2	0	0.2	2	12.4	20
5	0.2	0	0	0.2	2	12.6	20
10	0.2	0	0.2	0.3	3	16.3	30

We did this for 3 hours at 37℃, and then we did Purification. We got 30.7ng/µL GFP-DT.

Ligation

	torA	pSB1C3	pspA	DT	GFP-DT	Ligation High Ver.2	total
1	4	1	0	0	0	3	8
2	0	1	7	0	0	4	12
3	0	0	5	3	0	4	12
4	3	0	0	0	5	4	12

We did this for an hour at16℃.

March 22

PCR

We did PCR to amplify torA_signal that was product of PCR at March 5 with redesigned primers.

Buffer	dNTPs	MgSO4	Primer-F	Primer-R	Template	MilliQ	KOD plus neo	total
5	5	3	1.5	1.5	0.5	32.5	1	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles
→Purification 110.7ng/µL

Restriction

torA	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30

Ligation

	torA	pSSB1C3	pspA	DT	GFP-DT	Ligation high	total
1	4	3	0	0	0	4	11
2	3	0	0	0	3	3	9
3	0	0	5	5	0	5	15

torA (4µL)→512fmol
pSB1C3→54fmol
torA (3µL)→384fmol
GFP-DT→36fmol
pspA→377fmol
DT→65fmol

March 23

Screening PCR

torA (pSB1C3), torA-GFP-DT and pspA-DT

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

E.coli in liquid culture that had pspA(pSB1C3) also expressed RFP.

Restriction

pspA	EcoR1	Spe1	BufferM	BSA	MilliQ	total
5	0.3	0.3	3	0.3	21.1	30

And then we did ethanol precipitation

Ethanol precipitation

pspA 11.5ng/µL.

Miniprep

Lacp+pSB3C5-8 77.9µg/mL
Lacp+pSB3C5-10 69.0µg/mL
Kil+DT-4 58.0µg/mL
Kil+DT-7 15.2µg/mL

Restriction

Lacp+pSB3C5-8	Spe1	Pst1	Buffer2	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

Kil+DT-4	Xba1	Pst1	Buffer2	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1.5 hours
And then we did Gel extraction.

Gel Extraction

Lacp+pSB3C5-8 40.4µg/mL
Kil+DT-4 26.8µg/mL

March 26

Miniprep

torA(pSB1C3) 18.5[ng/µL]
torA-GFP-DT 20.0[ng/µL]

Restriction

torA(pSB1C3)	EcoR1	Pst1	BufferH	BSA	MilliQ	total
5	0.2	0.2	2	0.2	12.4	20
5	0.2	0	2	0.2	12.6	20

torA-GFP-DT	EcoR1	Xba1	Pst1	BufferH	BufferM	BSA	MilliQ	total
5	0.2	0	0.2	2	0	0.2	12.4	20
5	0.2	0	0	2	0	0.2	12.6	20
20	0	0.2	0.2	0	3	0.3	6.3	30

We did Gel extraction and then got ??? 28.7[ng/µL]

Ligation

Lacp (pSB3C5)	torA-GFP-DT	Ligation High Ver.2	total
1	5	3	9

Lacp : 22fmol
torA-GFP-DT : 197fmol

pspA	pSB1C3	Ligation High Ver.2	total
10	1	5	16

pspA	DT	Ligation High Ver.2	total
10	1	5	16

pspA : 180fmol
pSB1C3 : 18fmol
DT : 16fmol

LacP(pSB3C5)	Kil-DT	Ligation High Ver.2	total
1	5	3	9

for 2 hours at 16℃

Transformation

Lacp-Kil-DT	competent cell	total
1	10	11

March 27

Miniprep

We retryed miniprep of torA(pSB1C3).
We got torA and its concentration was 39.3[ng/µL].

Transformation

Name	Well	Sample	Competent Cells	Total	Plate	Colony
BBa_K117004	14J(2011 plate2)	5	20	?	?	?

We added 100[µL] of culture medium before we started culturing the E.coli.

Screening PCR

Quick Taq	VF2	VR	MilliQ	Total
25	1	1	23	50

Lacp-torA-GFP-DT 1, 3, 5, 6, 8, 9 was successful.
pspA-DT was failed.
E.coli that had pspA(pSB1C3) did not make any colony.
Lacp-Kil-DT 1,2,3,4,5,6,7,8 was failed.

Liquid culture

Lacp-torA-GFP-DT

@@ Line 4: / Line 4: @@
 <div class="_kyoto-note">
 ==February 7==
-'''Preculture'''   <small>by_???</small><br>
+====Preculture====   <small>by_???</small><br>
 We started preculture at 12:10.<br>
@@ Line 28: / Line 28: @@
 ==March 4==
-'''Sequence of tatABCD'''<br>
+====Sequence of tatABCD====
 {| class="wikitable" style="text-align: right;"
 !Quick Taq||primer-f||promer-r sequence||template||MilliQ||total
@@ Line 39: / Line 39: @@
 |25||1||1||1||23||50
 |}
-'''Colony PCR of TMAO'''<br>
+====Colony PCR of TMAO====
 {| class="wikitable" style="text-align: right;"
 !buffer||dNTPs||NgSO4||primer-f||primerr-r||KOD plus||MilliQ||total
@@ Line 52: / Line 52: @@
 ℃, 2.5min<br>
 →25cycles<br><br>
-'''Electrophoresis'''<br>
+====Electrophoresis====
 . 1kb ladder<br>
 . tatABCD1<br>
@@ Line 58: / Line 58: @@
 . TMAO<br>
 . 1kb ladder<br><br>
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !TMAO||EcoR1||BufferH||BSA||MilliQ||total
@@ Line 70: / Line 70: @@
 |}
 at 37℃ for 1 hour<br><br>
-'''Electrophoresis'''<br><br>
+====Electrophoresis====<br>
-'''Transformation'''<br>
+====Transformation====
 {|class="wikitable"
 !pSB1C3||competent cell(made at 2/8)||total
@@ Line 79: / Line 79: @@
 ==March 5==
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !pSB1C3(Xba1, Spe1)||Pst1||BufferH||BSA||MilliQ||total
@@ Line 91: / Line 91: @@
 at 37℃ for 1 hour<br>
 →Then we did ethanol precipitation<br><br>
-'''Ligation'''<br>
+====Ligation====
 {| class="wikitable"
 !Kil(EcoR1, Spe1)||pSB1C3(EcoR1)||Ligation High||total
@@ Line 98: / Line 98: @@
 |}
 at 16℃ for 1 hour<br><br>
-'''Transformation'''<br>
+====Transformation====
 {| class="wikitable"
 !Kil||competent cell||total
@@ Line 105: / Line 105: @@
 |}
 We used commercially available competent cells in this time.<br><br>
-'''PCR'''<br>
+====PCR====
 TMAO<br>
 {| class="wikitable"
@@ Line 112: / Line 112: @@
 |5||5||3||1.5||1.5||1||1||32||50
 |}
-'''Electrophoresis'''
+====Electrophoresis====
 (31)<br><br>
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 ! ||Lacp||pSB3C5||EcoR1||Pst1||BufferH||BSA||MilliQ||total
@@ Line 125: / Line 125: @@
 →Ethanol precipitation　45.4µg/mL<br>
 →Gel extraction　38.7µg/mL<br><br>
-'''Ligation'''<br>
+====Ligation====
 {| class="wikitable"
 !LacP||pSB3C5||Ligation High||total
@@ Line 132: / Line 132: @@
 |}
 at 4℃ for overnight<br><br>
-'''Transformation'''<br>
+====Transformation====
 {| class="wikitable"
 !Lacp+pSB3C5||competent cell||total
@@ Line 142: / Line 142: @@
 on ice for 2 mins.<br>
 After we incubate with 200µL of SOC culture for 1 hour, we did plating on LB culture with CP<br><br>
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !GFP Plasmid||EcoR1||Spe1||Buffer2||BSA||MilliQ||total
@@ Line 149: / Line 149: @@
 |}
 at 37℃ for 2 hours
-'''Electrophoresis'''<br>
+====Electrophoresis====
 . Ladder 2µL<br>
 . GFP Plasmid (already restricted) 2µL + Loading Dye 2µL + MilliQ 8µL<br>
 . Ladder 2µL<br>
-'''PCR'''<br>
+====PCR====
 torA signal and pspA
 pspAはコロニーPCR<br>
@@ Line 165: / Line 165: @@
 ℃, 30sec<br>
 ℃, 30sec<br>
-'''electrophoresis'''<br>
+====electrophoresis====
 . 100bp Ladder<br>
 . torA signal (272bp)<br>
@@ Line 172: / Line 172: @@
 ==March 6==
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !GFP||EcoR1||Spe1||BufferM||BSA||MilliQ||total
@@ Line 181: / Line 181: @@
 We did incubate at 37℃ for 1.5hours.<br>
 And we did gel extraction on 3/7 and get 40.0μg/mL GFP.<br><br>
-'''PCR'''<br>
+====PCR====
 {| class="wikitable"
 !buffer||dNTPs||MgSO4||Primer-f||Primer-r||template||KOD plus neo||MilliQ||total
@@ Line 192: / Line 192: @@
 ℃　(torA 10sec / pspA 30sec)     25 cycles<br>
 We used product of PCR on 3/5 of torA and pspA as template.<br><br>
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !Kil||EcoR1||Spe1||BufferM||BSA||MilliQ||total
@@ Line 205: / Line 205: @@
 at 37℃ for 2 hours<br>
 → purification 37.7ng/μL<br><br>
-'''Ligation'''<br>
+====Ligation====
 {| class="wikitable"
 !Kil||pSB1C3||Ligation High Ver.2||total
@@ Line 216: / Line 216: @@
 ==March 7==
-'''Electrophoresis'''[[File:Electrophoresis0307.JPG|400px|thumb|right]]<br>
+====Electrophoresis====[[File:Electrophoresis0307.JPG|400px|thumb|right]]<br>
 . 1kb ladder 2µL<br>
 . pspA (PCR product)2.5µL + Loading Dye 0.5µL<br>
@@ Line 222: / Line 222: @@
 . 1kb ladder 2µL<br>
 *The GFP was gel extracted on 3/6 and concentrated by Vacuum in 150µg/mL<br><br>
-'''Ligation'''<br>
+====Ligation====
 {| class="wikitable"
 !VectorDNA||GFP||Ligation High Ver.2||total
@@ Line 228: / Line 228: @@
 |5||15||10||30
 |}
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !torA||EcoR1||Spe1||bufferM||BSA||MilliQ||total
@@ Line 240: / Line 240: @@
 |}
 at 37℃ for 1.5 hours<br><br>
-'''Purification'''<br>
+====Purification====
 torA→31.8ng/µL<br>
 pspA→49.3ng/µL<br><br>
-'''Ligation'''<br>
+====Ligation====
 {| class="wikitable"
 !torA||pSB1C3||Ligation High Ver.2||total
@@ Line 261: / Line 261: @@
 ==March 8==
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !pSB4K5||EcoR1||Spe1||BufferM||BSA||MilliQ||total
@@ Line 270: / Line 270: @@
 at 37℃ for 1 hour.<br>
 →Purification : 36.6ng/µL<br><br>
-'''Ligation'''<br>
+====Ligation====
 {| class="wikitable"
 !Kil||pSB4K5||Ligation High Ver.2||total
@@ Line 279: / Line 279: @@
 *Kil→37.7ng/µL×10µL=377ng=879fmol<br>
 *pSB4K5→36.6ng/µL×1µL=36.6ng=86fmol<br><br>
-'''Liquid culture'''<br>
+====Liquid culture====
 Lacp + pSB3C5 -1, 2<br><br>
-'''Transformation'''<br>
+====Transformation====
 {| class="wikitable"
 !torA||pspA||competent cell||total
@@ Line 292: / Line 292: @@
 ==March 9==
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !tatABCD||Xba1||Pst1||BufferM||BSA||MilliQ||total
@@ Line 300: / Line 300: @@
 at 37℃ for 1hour<br>
 →purification : 75.0μg/mL    (1.11  260/280 , 0.81  260/230)<br><br>
-'''Miniprep'''<br>
+====Miniprep====
 lacP + pSB3C5 1    80.5μg/mL    (1.78  260/280 , 2.00  260/230)<br>
 lacP + pSB3C5 2    107.2μg/mL  (1.83  260/280 , 1.90  260/230)<br><br>
-'''Colony PCR'''<br>
+====Colony PCR====
 {| class="wikitable"
 !Quick Taq||VF||VR||MilliQ||total
@@ Line 314: / Line 314: @@
 ℃ 6sec<br>
 cycles<br><br>
-'''Ligation'''<br>
+====Ligation====
 {| class="wikitable"
 !tatABCD||constP J23107||Ligation High Ver.2||total
@@ Line 322: / Line 322: @@
 tatABCD : 227fmol<br>
 constP J23107 : 21fmol<br><br>
-'''Transformation'''
+====Transformation====
 {| class="wikitable"
 ! ||pspA||torA||Kil||competent cell
@@ Line 332: / Line 332: @@
 |3||0||0||1||10
 |}
-'''Miniprep'''<br>
+====Miniprep====
 mL of plusgrow which had been cultured for overnight.<br>
 pSB4K5 : 80.5μg/mL<br>
 ==March 10==
-'''Screening PCR'''<br>
+====Screening PCR====
 {| class="wikitable"
 !Quick Taq||VF||VR||MilliQ||total
@@ Line 351: / Line 351: @@
 , 100bp ladder<br>
 ,3,4, torA signal<br><br>
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !LacP-pSB3C5||Spe1||Pst1||BufferM||BSA||MilliQ||total
@@ Line 366: / Line 366: @@
 for 2.5 hours at 37℃<br>
 →purification  91.8ng/μL<br><br>
-'''Ligation'''<br>
+====Ligation====
 {| class="wikitable"
 !torA||Lacp-pSB3C5||Ligation High Ver.2||total
@@ Line 377: / Line 377: @@
 ==March 11==
-'''Miniprep'''<br>
+====Miniprep====
 We used 3μL of plus grow that we had cultured for overnight.<br>
 torA : 62.8ng/μL<br><br>
-'''Screening PCR'''<br>
+====Screening PCR====
 {| class="wikitable"
 !Quick Taq||VF||VR||MilliQ||total
@@ Line 386: / Line 386: @@
 |25||1||1||23||50
 |}
-'''Electrophoresis'''<br>
+====Electrophoresis====
 [[File:Electrophoresis031101.JPG|400px|thumb|right]]
 . 1kb ladder<br>
@@ Line 402: / Line 402: @@
 ==March 12==
-'''Transformation'''<br>
+====Transformation====
 {| class="wikitable"
 !DT(1ng/μL)||DT(0.1ng/μL)||Kil||lacP-torA||MilliQ||competent cell||total
@@ Line 416: / Line 416: @@
 |0||0||0||0||1||20||21
 |}
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !pspA||EcoR1||Spe1||BufferM||BSA||MilliQ||total
@@ Line 424: / Line 424: @@
 at 37℃ for 4 hours<br>
 → We did purification and got 48.3ng/μL pspA.<br><br>
-'''Ligation'''<br>
+====Ligation====
 {| class="wikitable"
 ! |pspA||pSB1C3||MilliQ||Ligation High Ver.2||total
@@ Line 436: / Line 436: @@
 ==March 13==
-'''Miniprep'''
+====Miniprep====
 pSB1C3  74.2µg/mL  1.65 (260/280)  1.31 (260/230)
 ==March 14==
-'''Restriction'''
+====Restriction====
 {| class="wikitable"
 !pSB1C3||EcoR1||Spe1||BufferM||BSA||MilliQ||total
@@ Line 446: / Line 446: @@
 |}
 We did gel extraction and got 47.2ng/µL pSB1C3 but we did not cut out RFP.<br><br>
-'''Ligation'''<br>
+====Ligation====
 {| class="wikitable"
 !torA||pSB1C3||Ligation High Ver.2||total
@@ Line 459: / Line 459: @@
 *torA : 0.707pmol<br>
 *pSB1C3 : 0.068pmol<br><br>
-'''Liquid culture'''<br>
+====Liquid culture====
 We cultured Lacp-pSB3C5 1,2,3,4,5 (CP tolerance)on culture with Amp.<br>
 →Only 4 which did not be cultured succeeded.
 ==March 15==
-'''Liquid culture'''<br>
+====Liquid culture====
 We cultured Lacp-pSB3C5 6,7,8,9,10 on culture with ampicillin.<br>
 →6,8,9,10 were succeeded.<br><br>
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !GFP||EcoR1||Spe1||Buffer2||BSA||MilliQ||total
@@ Line 479: / Line 479: @@
 |}
 for 2 hours at 37℃.<br><br>
-'''Miniprep'''<br>
+====Miniprep====
 pspA (pSB1C3)  40.5ng/µL<br><br>
-'''Ligation'''
+====Ligation====
 {| class="wikitable"
 !pspA||DT||Ligation High Ver.2||total
@@ Line 489: / Line 489: @@
 *pspA : 385fmol<br>
 *DT : 36fmol<br><br>
-'''Transformation'''
+====Transformation====
 {| class="wikitable"
 !J23107-tatABCD||DT (0.1ng/µL)||DT (0.01ng/µL)||pspA-DT||competent cells on 3/15||total
@@ Line 501: / Line 501: @@
 |0||0||0||2||20||22
 |}
-'''Screening PCR'''<br>
+====Screening PCR====
 Kil, pspA and torA<br>
 {| class="wikitable"
@@ Line 510: / Line 510: @@
 ==March 16==
-'''Miniprep'''<br>
+====Miniprep====
 torA (pSB1C3)  68.8ng/µL<br>
 Kil (pSB4K5)  92.7ng/µL<br>
 torA was red for some reason. We do not know why.<br><br>
-'''Colony PCR'''<br>
+====Colony PCR====
 {| class="wikitable"
 !Quick Taq||Primer-r||Primer-f||MilliQ||total
@@ Line 525: / Line 525: @@
 ℃, 6sec<br>
 →25cycles<br><br>
-'''Electrophoresis'''<br>
+====Electrophoresis====
 [[File:Electrophoresis0316.JPG|400px|thumb|right]]
 The results were shown as photograph in the right.<br><br>
-'''Checking Transformation Efficiency'''<br>
+====Checking Transformation Efficiency====
 competent cells that were made on March 15.<br>
 DNA : 0.02ng → 668 colonies  Transformation Efficiency : 3.3×10^7<br>
 DNA : 0.2ng → 1739 colonies  Transformation Efficiency : 8.7×10^6<br><br>
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !pSB1C3||EcoR1||Spe1||BSA||BufferM||BufferH||MilliQ||total
@@ Line 560: / Line 560: @@
 for overnight at 37℃.<br>
 We did this to confirm.<br><br>
-'''Ligation'''<br>
+====Ligation====
 {| class="wikitable"
 !torA||pSB1C3||Ligation High Ver.2||total
@@ Line 576: / Line 576: @@
 ==March 17==
-'''Miniprep'''
+====Miniprep====
 J23107-tatABCD  72.7ng/µL<br>
 pspA-DT  50.5ng/µL<br><br>
-'''Checking the Insert'''
+====Checking the Insert====
 {| class="wikitable"
 !J21037-tatABCD||EcoR1||Pst1||BSA||BufferH||MilliQ||total
@@ Line 600: / Line 600: @@
 ==March 19==
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !DT||EcoR1||Xba1||BSA||BufferM||MilliQ||total
@@ Line 607: / Line 607: @@
 |}
 We did Gel extraction and got 17.2ng/µL of DT.<br><br>
-'''Ligation'''<br>
+====Ligation====
 {| class="wikitable"
 !Kil||DT||Ligation High Ver.2||total
@@ Line 614: / Line 614: @@
 |}
 We did this for an hour at 16℃.<br><br>
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !GFP||EcoR1||Spe1||BSA||BufferM||MilliQ||total
@@ Line 621: / Line 621: @@
 |}
 We did this for 4 hours at 37℃<br><br>
-'''Ligation'''
+====Ligation====
 {| class="wikitable"
 !pspA||DT||Ligation High Ver.2||total
@@ Line 637: / Line 637: @@
 *pspA (4µL)→339fmol<br>
 *pSB1C3→34fmol<br><br>
-'''Transformation'''<br>
+====Transformation====
 pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT
 ==March 20==
-'''Screaning PCR'''<br>
+====Screaning PCR====
 {| class="wikitable"
 !Quick Taq||Primer-R||Primer-F||MilliQ||total
@@ Line 659: / Line 659: @@
 *pspA→○<br>
 *pspA-DT→×<br><br>
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !torA||EcoR1||Spe1||BSA||BufferM||MilliQ||total
@@ Line 675: / Line 675: @@
 ==March 21==
-'''Miniprep'''<br>
+====Miniprep====
 GFP-DT-1 : 77.7ng/µL<br>
 GFP-DT-2 : 67.6ng/µL<br><br>
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !pSB1C3||EcoR1||Spe1||BSA||BufferM||MilliQ||total
@@ Line 697: / Line 697: @@
 |}
 We did this for 3 hours at 37℃, and then we did Purification. We got 30.7ng/µL GFP-DT.<br><br>
-'''Ligation'''<br>
+====Ligation====
 {| class="wikitable"
 ! ||torA||pSB1C3||pspA||DT||GFP-DT||Ligation High Ver.2||total
@@ Line 712: / Line 712: @@
 ==March 22==
-'''PCR'''<br>
+====PCR====
 We did PCR to amplify torA_signal that was product of PCR at March 5 with redesigned primers.<br>
 {| class="wikitable"
@@ Line 725: / Line 725: @@
 →30cycles<br>
 →Purification  110.7ng/µL<br><br>
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !torA||EcoR1||Spe1||BSA||BufferM||MilliQ||total
@@ Line 731: / Line 731: @@
 |10||0.2||0.2||0.3||3||16.3||30
 |}
-'''Ligation'''
+====Ligation====
 {| class="wikitable"
 ! ||torA||pSSB1C3||pspA||DT||GFP-DT||Ligation high||total
@@ Line 749: / Line 749: @@
 ==March 23==
-'''Screening PCR'''<br>
+====Screening PCR====
 torA (pSB1C3), torA-GFP-DT and pspA-DT<br>
 {| class="wikitable"
@@ Line 757: / Line 757: @@
 |}
 E.coli in liquid culture that had pspA(pSB1C3) also expressed RFP.<br><br>
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !pspA||EcoR1||Spe1||BufferM||BSA||MilliQ||total
@@ Line 764: / Line 764: @@
 |}
 And then we did ethanol precipitation<br><br>
-'''Ethanol precipitation'''<br>
+====Ethanol precipitation====
 pspA 11.5ng/µL.<br><br>
-'''Miniprep'''<br>
+====Miniprep====
 Lacp+pSB3C5-8   77.9µg/mL<br>
 Lacp+pSB3C5-10   69.0µg/mL<br>
 Kil+DT-4   58.0µg/mL<br>
 Kil+DT-7   15.2µg/mL<br><br>
-'''Restriction'''<br>
+====Restriction====
 {| class="wikitable"
 !Lacp+pSB3C5-8||Spe1||Pst1||Buffer2||BSA||MilliQ||total
@@ Line 784: / Line 784: @@
 at 37℃ for 1.5 hours<br>
 And then we did Gel extraction.<br><br>
-'''Gel Extraction'''<br>
+====Gel Extraction====
 Lacp+pSB3C5-8   40.4µg/mL<br>
 Kil+DT-4   26.8µg/mL<br>
 ==March 26==
-'''Miniprep'''<br>
+====Miniprep====
 torA(pSB1C3) 18.5[ng/µL]<br>
 torA-GFP-DT 20.0[ng/µL]<br><br>
-'''Restriction'''
+====Restriction====
 {| class="wikitable"
 !torA(pSB1C3)||EcoR1||Pst1||BufferH||BSA||MilliQ||total
@@ Line 810: / Line 810: @@
 |}
 We did Gel extraction and then got ??? 28.7[ng/µL]<br><br>
-'''Ligation'''
+====Ligation====
 {| class="wikitable"
 !Lacp (pSB3C5)||torA-GFP-DT||Ligation High Ver.2||total
@@ Line 837: / Line 837: @@
 |}
 for 2 hours at 16℃<br><br>
-'''Transformation'''<br>
+====Transformation====
 {| class="wikitable"
 !Lacp-Kil-DT||competent cell||total
@@ Line 845: / Line 845: @@
 ==March 27==
-'''Miniprep'''<br>
+====Miniprep====
 We retryed miniprep of torA(pSB1C3).<br>
 We got torA and its concentration was 39.3[ng/µL].<br><br>
-'''Transformation'''<br>
+====Transformation====
 {| class="wikitable"
 !Name||Well||Sample||Competent Cells||Total||Plate||Colony
@@ Line 855: / Line 855: @@
 |}
 We added 100[µL] of culture medium before we started culturing the E.coli.<br><br>
-'''Screening PCR'''<br>
+====Screening PCR====
 {| class="wikitable"
 !Quick Taq||VF2||VR||MilliQ||Total
@@ Line 865: / Line 865: @@
 E.coli that had pspA(pSB1C3) did not make any colony.<br>
 Lacp-Kil-DT 1,2,3,4,5,6,7,8 was failed.<br><br>
-'''Liquid culture'''<br>
+====Liquid culture====
 Lacp-torA-GFP-DT<br>
 </div>
 {{Kyoto/footer}}

Team:Kyoto/Secretion/Notebook

From 2012.igem.org

Revision as of 16:31, 9 September 2012

Contents

Secretion Notebook

February 7

February 8

March 1

March 2

March 3

PCR

Miniprep

March 4

Sequence of tatABCD

Colony PCR of TMAO

Electrophoresis

Restriction

Transformation

March 5

Restriction

Ligation

Transformation

PCR

Electrophoresis

Restriction

Ligation

Transformation

Restriction

Electrophoresis

PCR

electrophoresis

March 6

Restriction

PCR

Restriction

Ligation

March 7

Ligation

Restriction

Purification

Ligation

March 8

Restriction

Ligation

Liquid culture

Transformation

March 9

Restriction

Miniprep

Colony PCR

Ligation

Transformation

Miniprep

March 10

Screening PCR

Restriction

Ligation

March 11

Miniprep

Screening PCR

Electrophoresis

March 12

Transformation

Restriction

Ligation

March 13

Miniprep

March 14

Restriction

Ligation

Liquid culture

March 15

Liquid culture

Restriction

Miniprep

Ligation

Transformation

Screening PCR

March 16

Miniprep