Difference between revisions of "Team:Kyoto/Notebook"

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<p class="keln_note">PCR<br>98℃ 30s<br>98℃* 10s<br>65℃* 30s<br>72℃* 1min<br>72℃ 2min<br>4℃ ∞<br> (*× 30 cycle)</p>
 
<p class="keln_note">PCR<br>98℃ 30s<br>98℃* 10s<br>65℃* 30s<br>72℃* 1min<br>72℃ 2min<br>4℃ ∞<br> (*× 30 cycle)</p>
 +
<!------------ Table END ------------>
 +
<!-- Table Generated by KELN Author: Nakazato -->
 +
<div class="keln_container">
 +
<a name="0831" class = "kyoto-jump"></a>
 +
<span class="keln_date"><h3>8/31</h3></span>
 +
<span class="keln_exp"><h4>OE-PCR</h4></span>
 +
<span class="keln_researcher"></span>
 +
<table class="keln_table">
 +
<tr><th>sample</th><th>template</th><th>volume (ul)</th><th>5× Q5 Reaction Buffer (ul)</th><th>10uM dNTPs (ul)</th><th>10uM Z026℗</th><th>10uM Z027℗</th><th>Z026-Z039 (ul)</th><th>Z040-Z027 (ul)</th><th>Q5 DNA Polymerase (ul)</th><th>DW (ul)</th><th>Total (ul)</th></tr>
 +
<tr><td>A</td><td>AtHKT1</td><td>5</td><td>10</td><td>1</td><td>2.5</td><td>2.5</td><td>5</td><td>5</td><td>0.5</td><td>18.5</td><td>50</td></tr>
 +
<tr><td>B</td><td>AtNHXS1</td><td>5</td><td>10</td><td>1</td><td>2.5</td><td>2.5</td><td>5</td><td>5</td><td>0.5</td><td>18.5</td><td>50</td></tr>
 +
<tr><td>C</td><td>GFP</td><td>5</td><td>10</td><td>1</td><td>2.5</td><td>2.5</td><td>5</td><td>5</td><td>0.5</td><td>18.5</td><td>50</td></tr>
 +
<tr><td>D</td><td>RFP</td><td>5</td><td>10</td><td>1</td><td>2.5</td><td>2.5</td><td>5</td><td>5</td><td>0.5</td><td>18.5</td><td>50</td></tr>
 +
</table>
 +
</div>
 +
<p class="keln_note">PCR<br>98℃ 30s<br>98℃* 10s<br>55℃* 30s<br>72℃* 1m30s<br>72℃ 2min<br>4℃ ∞<br> (*× 30 cycle)</p>
 
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Revision as of 12:55, 10 September 2018

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
componentvolume(μl)
BSA0.5
buffer(10xH)5
EcoR10.5
Pst10.5
Dpn10.5
dH2O18

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
samplePrimer Forwardvolume (ul)Primer Reversevolume (ul)template DNAvolume (ul)Q5 HF 2xMasterMix (ul)DW (ul)
1Z0071.25Z0091.25Yeast genome1.01.09.0
2Z0081.25Z0111.25GFP1.01.09.0
3Z0101.25Z0121.25Yeast genome1.01.09.0
4Z0071.25Z0141.25Yeast genome1.01.09.0
5Z0131.25Z0161.25RFP1.01.09.0
6Z0151.25Z0121.25Yeast genome1.01.09.0

7/6

OE-PCR

 Nambu, Shimazoe
samplePrimer Forwardvolume (ul)Primer Reversevolume (ul)Template DNAvolume (ul)Q5 HF2x Master Mix (ul)DW (ul)
1Z0071.25Z0091.25Yeast genome1.012.59.0
2Z0101.25Z0121.25Yeast genome1.012.59.0
3Z0071.25Z0141.25Yeast genome1.012.59.0
4Z0151.25Z0121.25Yeast genome1.012.59.0
5*Z0071.25Z0091.25p0791.012.59.0
6*Z0101.25Z0121.25p0791.012.59.0
7*Z0071.25Z0141.25p0791.012.59.0
8*Z0151.25Z0121.25p0791.012.59.0

*sample5~8 x 2set

7/6

colony_PCR
Mangrin colonyDW (ul)Q5 HF2x MasterMix (ul)m13 Forward (ul)m13 Reverse (ul)
12.02.50.250.25
22.02.50.250.25
32.02.50.250.25
42.02.50.250.25
52.02.50.250.25

Store PCR product at 4℃.

7/10

Restriction Enzyme Digestion

 Tong, Tamukai
componentvolume(μl)
BSA0.5
buffer(10xH)5
EcoR10.5
Pst10.5
Dpn10.5
dH2O18

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
sampleKOD one PCR master mix (ul)TDH promotervolume (ul)ORFvolume (ul)CYC terminatorvolume (ul)DW (ul)
A12.551.5GFP1.561.58.0
B12.571.5RFP1.581.58.0
C12.551.5GFP1.561.58.0
D12.571.5RFP1.581.58.0

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
ComponentVolume(μl)
DW30
Q537.5
m13f3.75
m13r3.75
colonyPoke
Total75

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

8/7

YPD

 Tong
Component(1L)(900mL)(600mL)
Peptone20g18g12g
Yeast Extract10g9g6g
Agar15g13.5g9g
DWxmL810ml480mL
Glucose+DW20g+ymL (x+y=1000)18g12g

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
AOD
5620.38
5610.40
5640.36
5540.33
5550.38
5470.30
5570.32
5590.39

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
Number123456
vector426316426316426316
insertMangrinMangrinZrFPS1-2ZrFPS1-2McHKT2McHKT2

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
Tryptone8g
Yeast Extract4g
Agar8g
Glucose8g
NaCl100g

Normalnull
Tryptone4g
Yeast Extract2g
Agar4g
Glucose4g

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

8/14

Transformation

 Tamukai
Sample NameSample Volume (&microl)Competent Cells/(&microl)
Mangrin315
ZrFPS1-2315
McHKT2315

8/18

Colony PCR

 Nambu
ComponentAmount(&microl)
DW112
Q5140
m13f14
m13r14
ColonyPoke
Total280

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
MaterialVolume
LB90ml
Amp90ul

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
Number123456
gBlockMangrinZrFPS1-2ndMcHKT2AtHKT1ZrFPS1-1stAtNHXS1

mix the following material

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

putin HeatBlock overnight at 18℃

8/14gel

Transformation
See "Protocol" Page

8/19

PCR

 Nambu
samplecontent (gBlock)primer1primer2
1MangrinZ026Z027
2ZrFPS1_2ndZ026Z027
3McHKT2Z026Z027
4AtHKT1Z026Z027
5ZrFPS1_1stZ026Z027
6AtNHXS1Z026Z027
7SseNHX1Z026Z027
8AVP1_1st + AVP1_2ndZ026Z027

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

8/20

Ligation

 Nambu
numbergBlock (Insert)volume (ul)SmaCIP (backbone) (ul)Ligation High (ul)
7SdrG112
8SdrG-Fg overlap112
9ZrGPD1112

Put in Incubator overnight at 18℃.

8/20

Transformation

 Nambu
Sample NameSample Volume (&microl)Competent Cells/(&microl)Medium
Mangrin416SOC Medium
ZrFPS1_2nd416SOC Medium
McHKT2416SOC Medium
AtHKT1416SOC Medium
ZrFPS1_1st416SOC Medium
AtNHXS1416SOC Medium

8/21

Restriction Enzyme Digestion

 Shimazoe
plasmid plasmid volume (ul)DW (ul)10x Fast digest buffer (ul)EcoR1 (ul)Pst1 (ul)Total (ul)
1721421120
1911421120
2621421120
11021421120
12021421120

8/21

Digest plasmid

 Tong
plasmidplasmid volume (ul)DW (ul)10x Fast Digest Buffer (ul)EcoR1 (ul)Pst1 (ul)
304214211
309214211
313214211
314214211
317214211
325214211
342214211
322214211
336214211
343214211
352214211
353214211
354214211

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)0.53.55Z0640.5Z026(p)0.5
83 (AVP1_2nd)0.53.55Z0650.5Z027(p)0.5

8/25

colony_PCR

 Shimazoe
sampleQ5 (ul)DW (ul)primer m13f (ul)primer m13r (ul)
382.52.00.250.25
832.52.00.250.25

colony PCR x 2 set

8/27

OE-PCR

 Shimazoe
contentvolume (ul)
DW19
KOD one25
primer Z0261.5
prmer Z0271.5
AVP1-11
AVP1-22

8/26

Liquid Culture

 Nambu
sampleLB (ml)Amp(100mg/ml) (ul)
5055050
5065050
5075050
5085050
5165050
5225050
5305050

Put them in incubator at 37℃ for 19 hours.

8/27

Miniprep_Nanodrop

 Nambu

sample:505,506,507,508,516,522,530

8/28

PCR

 Shimazoe
ComponentAmount(&microl)
DW8.7
KOD10
Z026℗0.6
Z0640.6
38(template)0.1
Total20

PCR
98℃* 10s
50℃* 5s
68℃* 20s
68℃ 1min
4℃ ∞
　(*× 40 cycle)

8/28

PCR

 Shimazoe
ComponentAmount(&microl)
DW8.7
KOD10
Z027℗0.6
Z0650.6
83(template)0.1
Total20

PCR
98℃* 10s
50℃* 5s
68℃* 20s
68℃ 1min
4℃ ∞
　(*× 40 cycle)

8/28

OE-PCR

 Shimazoe
ComponentAmount(&microl)
DW20
Z026℗1.5
Z027℗1.5
KOD one25
AVP1-1st1
AVP1-2nd1
Total50

PCR
98℃* 10s
65℃* 5s
68℃* 30s
68℃ 1min
4℃ ∞
　(*× 25 cycle )

8/28

Colony PCR

 Shimazoe
ComponentAmount(&microl)
2× Gflex PCR Buffer162.5
primer m13f6.5
primer m13r6.5
Tks Gflex DNA polymerase6.5
DW143
ColonyPoke
Total325

PCR
　　　　　　　　　 　　　　 　　　 number
94℃ 1min　　　　　　 SdrG　　　　 601~611
98℃* 10s　　　　　　Sdr-Fg　　　　 612~623
55℃* 15s　　　　　　ZrGPD1　　　 624~631
68℃* 2min　　　　　 Mangrin　　　 633~638
4℃ ∞ 　　　　　　　Zr-FPS1-2nd 　　 639~642
　(*× 30 cycle)　　　　McHKT2 　　　643~649
　　　　　　　　　　ZrFPS1-1st 　　 650~657

8/29

PCR

 Shimazoe
ComponentAmount(&microl)
5× Q5 Reaction Buffer15
10mM dNTPs1.5
10uM Z0413.75
10uM Z0423.75
TemplatePoke
Q5 DNA Polymerase0.75
DW47.25
Total72

Template
AtHKT1(304), SseNHXS1(317), AtNHXS1(336)

PCR
98℃ 30s
98℃* 10s
65℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)

8/29

PCR

 Shimazoe
ComponentAmount(&microl)
5× Q5 Reaction Buffer5
10mM dNTPs0.5
10uM Z0411.25
10uM Z0421.25
TemplatePoke
Q5 DNA Polymerase0.25
DW16.75
Total24

Template
FgβF3

PCR
98℃ 30s
98℃* 10s
65℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)

8/30

Reaction Enzyme Digestion

 Shimazoe
plasmidplasmid(ul)10× T(ul)10× BSA(ul)SmaⅠ(ul)CIAP(ul)DW(ul)Total/(ul)
4261010104264100
3161010104264100
4251010104264100
3151010104264100
4231010104264100
3131010104264100

6 sample
37℃ overnight

8/30

OE-PCR

 Shimazoe
numbersamplevolume (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)10uM Z026℗ (ul)10uM Z027℗ (ul)Z026-Z039 (ul)Z040-Z027 (ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
AAtHKT10.520.20.50.50.50.50.15.210
BSseNHX10.520.20.50.50.50.50.15.210
CAtNHXS10.520.20.50.50.50.50.15.210

PCR

98℃ 30s
98℃* 10s
72℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 25 cycle)

8/30

PCR

 Shimazoe
templatevolume (ul)primer①volume (ul)primer②volume (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
AVP1-1st0.510uM Z026℗0.510uM Z0640.520.20.16.210
AVP1-2nd0.510uM Z027℗0.510uM Z0650.520.20.16.210

PCR
98℃ 30s
98℃* 10s
67℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 25 cycle)

8/30

OE-PCR

 Shimazoe
numbertemplatevolume (ul)10uM Z026℗10uM Z027℗Z026-Z039 (ul)Z040-Z027 (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
AAtHKT110.50.51120.20.13.710
BSseNHX110.50.51120.20.13.710
CAtNHXS110.50.51120.20.13.710
DGFP10.50.51120.20.13.710
ERFP10.50.51120.20.13.710

PCR
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)

8/30

PCR

 Shimazoe
templatevolume (ul)10uM primer①volume (ul)10uM primer②volume (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
AVP1-1st (PCR products)1Z026℗1.25Z0641.2550.50.2515.7525
AVP1-1st (Plasmid)1Z026℗1.25Z0641.2550.50.2515.7525
AVP1-2nd (PCR products)1Z027℗1.25Z0651.2550.50.2515.7525
AVP1-2nd (Plasmid)1Z027℗1.25Z0651.2550.50.2515.7525

PCR
98℃ 30s
98℃* 10s
65℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)

8/31

OE-PCR
sampletemplatevolume (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)10uM Z026℗10uM Z027℗Z026-Z039 (ul)Z040-Z027 (ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
AAtHKT151012.52.5550.518.550
BAtNHXS151012.52.5550.518.550
CGFP51012.52.5550.518.550
DRFP51012.52.5550.518.550

PCR
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)