Difference between revisions of "Team:Kyoto/Notebook"

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<p class="keln_note">Store PCR product at 4℃.</p>
 
<p class="keln_note">Store PCR product at 4℃.</p>
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<!-- Table Generated by KELN Author: Tsuzuki -->
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<div class="keln_container">
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<a name="0712" class = "kyoto-jump"></a>
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<span class="keln_date"><h3>7/12</h3></span>
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<span class="keln_exp"><h4>OE~PCR</h4></span>
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<span class="keln_researcher">Nambu</span>
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<table class="keln_table">
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<tr><th>sample</th><th>KOD one PCR master mix (ul)</th><th>TDH promoter</th><th>volume (ul)</th><th>ORF</th><th>volume (ul)</th><th>CYC terminator</th><th>volume (ul)</th><th>DW (ul)</th></tr>
 +
<tr><td>A</td><td>12.5</td><td>5</td><td>1.5</td><td>GFP</td><td>1.5</td><td>6</td><td>1.5</td><td>8.0</td></tr>
 +
<tr><td>B</td><td>12.5</td><td>7</td><td>1.5</td><td>RFP</td><td>1.5</td><td>8</td><td>1.5</td><td>8.0</td></tr>
 +
<tr><td>C</td><td>12.5</td><td>5</td><td>1.5</td><td>GFP</td><td>1.5</td><td>6</td><td>1.5</td><td>8.0</td></tr>
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<tr><td>D</td><td>12.5</td><td>7</td><td>1.5</td><td>RFP</td><td>1.5</td><td>8</td><td>1.5</td><td>8.0</td></tr>
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</table>
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</div>
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<p class="keln_note">PCR <br>98℃　0:30<br>98℃*  0:10<br>45℃*  0:05<br>68℃*  0:05<br>68℃    2:00<br>10℃ <br>(* x 30 cycle)      </p>
 
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Revision as of 11:17, 5 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(&microl)
BSA0.5
buffer(10xH)5
EcoR10.5
Pst10.5
Dpn10.5
dH2O18

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

7/12

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)