Team:Kyoto/Notebook

• 
• Project
  • Description
  • Design
  　　　　
  • Results
  　　　　　　　　　
  • Demonstration
  • Discussion
• Modeling
• Lab Work
  • Notebook
  • Materials&Methods
  • Special Methods
  • Safety
• Parts
  • Basic Parts
  • Composite Parts
  • Improving parts
  • Parts Collection
• Human Practices
  • Human Practice/Silver
  • Integrated Human Practice/Gold
  • Collaborations
• Team
  • Members
  • Attributions

Notebook

July
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	31

August
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	31

September
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	31

October
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	31

7/3

Restriction Enzyme Digestion

Tong, Tamukai

component	volume(μl)
BSA	0.5
buffer(10xH)	5
EcoR1	0.5
Pst1	0.5
Dpn1	0.5
dH2O	18

add 4μl linearized plasmid backbone (25ng/μl)
add 4μl Enzyme Master Mix
digest 37℃, 30min
add 8μl NaOAc (pH5.2,3M)
add 16μl 99.5% EtOh
Vortex 2sec
add 184μl 99.5% EtOH
add 72μl DW

7/5

OE-PCR

Nambu, Shimazoe

sample	Primer Forward	volume (ul)	Primer Reverse	volume (ul)	template DNA	volume (ul)	Q5 HF 2xMasterMix (ul)	DW (ul)
1	Z007	1.25	Z009	1.25	Yeast genome	1.0	1.0	9.0
2	Z008	1.25	Z011	1.25	GFP	1.0	1.0	9.0
3	Z010	1.25	Z012	1.25	Yeast genome	1.0	1.0	9.0
4	Z007	1.25	Z014	1.25	Yeast genome	1.0	1.0	9.0
5	Z013	1.25	Z016	1.25	RFP	1.0	1.0	9.0
6	Z015	1.25	Z012	1.25	Yeast genome	1.0	1.0	9.0

7/6

OE-PCR

Nambu, Shimazoe

sample	Primer Forward	volume (ul)	Primer Reverse	volume (ul)	Template DNA	volume (ul)	Q5 HF2x Master Mix (ul)	DW (ul)
1	Z007	1.25	Z009	1.25	Yeast genome	1.0	12.5	9.0
2	Z010	1.25	Z012	1.25	Yeast genome	1.0	12.5	9.0
3	Z007	1.25	Z014	1.25	Yeast genome	1.0	12.5	9.0
4	Z015	1.25	Z012	1.25	Yeast genome	1.0	12.5	9.0
5*	Z007	1.25	Z009	1.25	p079	1.0	12.5	9.0
6*	Z010	1.25	Z012	1.25	p079	1.0	12.5	9.0
7*	Z007	1.25	Z014	1.25	p079	1.0	12.5	9.0
8*	Z015	1.25	Z012	1.25	p079	1.0	12.5	9.0

*sample5~8 x 2set

7/6

colony_PCR

Mangrin colony	DW (ul)	Q5 HF2x MasterMix (ul)	m13 Forward (ul)	m13 Reverse (ul)
1	2.0	2.5	0.25	0.25
2	2.0	2.5	0.25	0.25
3	2.0	2.5	0.25	0.25
4	2.0	2.5	0.25	0.25
5	2.0	2.5	0.25	0.25

Store PCR product at 4℃.

7/10

Restriction Enzyme Digestion

Tong, Tamukai

component	volume(μl)
BSA	0.5
buffer(10xH)	5
EcoR1	0.5
Pst1	0.5
Dpn1	0.5
dH2O	18

add 4, 8 μl (add two by two to sample)
add 4, 8μl Enzyme master mix (add two by two to sample)digest 37℃ 30min
70℃ 20min
add 1,1.6 μl NaOAc pH5.2, 3M(add two by two to sample)
20, 40μl (add two by two to sample) 99.5% EtOHVortex
preserve in -20℃

7/12

OE-PCR

Nambu

sample	KOD one PCR master mix (ul)	TDH promoter	volume (ul)	ORF	volume (ul)	CYC terminator	volume (ul)	DW (ul)
A	12.5	5	1.5	GFP	1.5	6	1.5	8.0
B	12.5	7	1.5	RFP	1.5	8	1.5	8.0
C	12.5	5	1.5	GFP	1.5	6	1.5	8.0
D	12.5	7	1.5	RFP	1.5	8	1.5	8.0

PCR
98℃　0:30
98℃* 0:10
45℃* 0:05
68℃* 0:05
68℃ 2:00
10℃
(* x 30 cycle)

7/31

Transformation & Blue-White Selection

Shimazoe

Mix 10μl of Competent Cell and 2μl G,R Ligation Product on ice
On ice for 30min
Heat Shock 42℃ for 45sec
On ice for 1,2 min
Add 100μl SOC medium and culture in 37℃ for 1h
Spread on a plate (50μl X-gal added)

8/1

Colony PCR

Tong

Component	Volume(μl)
DW	30
Q5	37.5
m13f	3.75
m13r	3.75
colony	Poke
Total	75

Add 0.6μl 10x Buffer to all sample (1~14)
Culture in 10mL

8/7

YPD

Tong

Component	(1L)	(900mL)	(600mL)
Peptone	20g	18g	12g
Yeast Extract	10g	9g	6g
Agar	15g	13.5g	9g
DW	xmL	810ml	480mL
Glucose+DW	20g+ymL (x+y=1000)	18g	12g

fluid culture
cultured 9 yeast strains in liquid YPD.
Put 15 ml of YPD medium in a 50 ml sterile tube
push the yeast with a white chip, put it in the tube
loosen the lid and put it in the shaker

8/8

OD measure

Tong

A	OD
562	0.38
561	0.40
564	0.36
554	0.33
555	0.38
547	0.30
557	0.32
559

For each 8 samples, transferred to a 1.5 ml tube　1mL each.
For soy sauce yeast only 15 ml was centrifuged, decantation was repeated and scaled down to 1 ml.

8/13

Ligation

Nambu

Number	1	2	3	4	5	6
vector	426	316	426	316	426	316
insert	Mangrin	Mangrin	ZrFPS1-2	ZrFPS1-2	McHKT2	McHKT2

Mix the following material (Contamination are shown in the table above)

Plasmid(vector): 0.5&microL
Insert: 1.0&microl
Ligation High: 1.5&microl

Put in Heatblock

8/13

YPD culture

Tong

NaCl 25%(400mL)	null
Tryptone	8g
Yeast Extract	4g
Agar	8g
Glucose	8g
NaCl	100g

Normal	null
Tryptone	4g
Yeast Extract	2g
Agar	4g
Glucose	4g

Make
25% NaCl YPD solid medium 400 mL
Normal YPD solid medium 200 mL
YPD liquid medium 500 mL

8/14

Transformation

Tamukai

Sample Name	Sample Volume (&microl)	Competent Cells/(&microl)
Mangrin	3	15
ZrFPS1-2	3	15
McHKT2	3	15

8/18

Colony PCR

Nambu

Component	Amount(&microl)
DW	112
Q5	140
m13f	14
m13r	14
Colony	Poke
Total	280

PCR　　　　　　　Colony
98℃ 0:30　　　　　 number　　Backbone　　Insert
98℃* 0:10　　　　　301~306　　SmaCIP　　AtHKT1
57℃* 0:30　　　　　307~317　　SmaCIP　　SseNHX1
72℃* 1:00　　　　　318~326　　SmaCIP　　ZrFPs1-1st
72℃ 2:00　　　　　 327~337　　SmaCIP　　AtNHXS1
4℃ ∞　　　　　　　338~356　　SmaCIP　　 FgβF3
(*× 30 cycle)

8/18

Liquid Culture

Nambu

Material	Volume
LB	90ml
Amp	90ul

mix materials ( Finally concentration Amp 100 ug/ml )
put colony* into the liquid
(*colony number: 304,309,310,313,314,315,317,322,325,336,342,343,352,353,354)
put in incubator at 37℃ for 18 hours

8/19

Ligation

Nambu

Number	1	2	3	4	5	6
gBlock	Mangrin	ZrFPS1-2nd	McHKT2	AtHKT1	ZrFPS1-1st	AtNHXS1

mix the following material

SmaCIP (Backbone) : 1ul
gBlock (Insert) : 1ul
Ligation High : 2ul
(Total : 4ul)

putin HeatBlock overnight at 18℃

Transformation
See "Protocol" Page

8/19

PCR

Nambu

sample	content (gBlock)	primer1	primer2
1	Mangrin	Z026	Z027
2	ZrFPS1_2nd	Z026	Z027
3	McHKT2	Z026	Z027
4	AtHKT1	Z026	Z027
5	ZrFPS1_1st	Z026	Z027
6	AtNHXS1	Z026	Z027
7	SseNHX1	Z026	Z027
8	AVP1_1st + AVP1_2nd	Z026	Z027

94℃ 2:00
98℃* 0:10
48℃ *0:30
68℃ *3:00
68℃ 0:10
10℃
* x 30 cycle

8/20

Ligation

Nambu

number	gBlock (Insert)	volume (ul)	SmaCIP (backbone) (ul)	Ligation High (ul)
7	SdrG	1	1	2
8	SdrG-Fg overlap	1	1	2
9	ZrGPD1	1	1	2

Put in Incubator overnight at 18℃.

8/20

Transformation

Nambu

Sample Name	Sample Volume (&microl)	Competent Cells/(&microl)	Medium
Mangrin	4	16	SOC Medium
ZrFPS1_2nd	4	16	SOC Medium
McHKT2	4	16	SOC Medium
AtHKT1	4	16	SOC Medium
ZrFPS1_1st	4	16	SOC Medium
AtNHXS1	4	16	SOC Medium

8/21

Restriction Enzyme Digestion

Shimazoe

plasmid	plasmid volume (ul)	DW (ul)	10x Fast digest buffer (ul)	EcoR1 (ul)	Pst1 (ul)	Total (ul)
17	2	14	2	1	1	20
19	1	14	2	1	1	20
26	2	14	2	1	1	20
110	2	14	2	1	1	20
120	2	14	2	1	1	20

8/21

Digest plasmid

Tong

plasmid	plasmid volume (ul)	DW (ul)	10x Fast Digest Buffer (ul)	EcoR1 (ul)	Pst1 (ul)
304	2	14	2	1	1
309	2	14	2	1	1
313	2	14	2	1	1
314	2	14	2	1	1
317	2	14	2	1	1
325	2	14	2	1	1
342	2	14	2	1	1
322	2	14	2	1	1
336	2	14	2	1	1
343	2	14	2	1	1
352	2	14	2	1	1
353	2	14	2	1	1
354	2	14	2	1	1

15min 37℃ incubate

8/25

OE-PCR

Shimazoe

plasmid	plasmid volume (ul)	DW (ul)	Q5 (ul)	primer1	primer1 volume (ul)	primer2	primer2 volume (ul)
38 (AVP1_1st)	0.5	3.5	5	Z064	0.5	Z026(p)	0.5
83 (AVP1_2nd)	0.5	3.5	5	Z065	0.5	Z027(p)	0.5

8/25

colony_PCR

Shimazoe

sample	Q5 (ul)	DW (ul)	primer m13f (ul)	primer m13r (ul)
38	2.5	2.0	0.25	0.25
83	2.5	2.0	0.25	0.25

colony PCR x 2 set

8/27

OE-PCR

Shimazoe

content	volume (ul)
DW	19
KOD one	25
primer Z026	1.5
prmer Z027	1.5
AVP1-1	1
AVP1-2	2