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Team:UT-Tokyo/Counter/Project/Lab
From 2014.igem.org
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<!-- Part Table --> | <!-- Part Table --> | ||
<FORM action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2014&group=UT-Tokyo" enctype="multipart/form-data" method="post" abp="163"> | <FORM action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2014&group=UT-Tokyo" enctype="multipart/form-data" method="post" abp="163"> | ||
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Revision as of 17:18, 17 October 2014
| Name | Type | Description | Designer | Length | |||
|---|---|---|---|---|---|---|---|
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461000" abp="49">BBa_K1461000</A> | RBS | crRBS | Takefumi Yoshikawa | 52 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461001" abp="64">BBa_K1461001</A> | Regulatory | ecf20 promoter | Takefumi Yoshikawa | 80 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461002" abp="79">BBa_K1461002</A> | Regulatory | ecf11 promoter | Takefumi Yoshikawa | 80 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461003" abp="94">BBa_K1461003</A> | RNA | taRNA | Takefumi Yoshikawa | 71 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461004" abp="109">BBa_K1461004</A> | Coding | ecf20 | Takefumi Yoshikawa | 582 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461005" abp="124">BBa_K1461005</A> | Coding | ecf11 | Takefumi Yoshikawa | 609 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461006" abp="139">BBa_K1461006</A> | Coding | anti ecf20 | Takefumi Yoshikawa | 432 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461007" abp="154">BBa_K1461007</A> | Coding | anti ecf11 | Takefumi Yoshikawa | 663 |
<FORM action="/cgi/partsdb/pgroup.cgi?pgroup=iGEM2014&group=UT-Tokyo" enctype="multipart/form-data" method="post" abp="163">
| UT-Tokyo 2014 iGEM Team Parts Sandbox | <A href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2014&group=UT-Tokyo&Editing=Editing" abp="171">Edit</A> |
</FORM>
| Name | Type | Description | Designer | Length | |||
|---|---|---|---|---|---|---|---|
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461008" abp="194">BBa_K1461008</A> | Coding | GFP (AAV tag +) | Takefumi Yoshikawa | 742 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461009" abp="209">BBa_K1461009</A> | Coding | GFP (AAV tag +) | Takefumi Yoshikawa | 742 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461200" abp="329">BBa_K1461200</A> | Composite | taRNA (+d.term) | Takefumi Yoshikawa | 208 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461201" abp="344">BBa_K1461201</A> | Composite | ecf20 (+RBS, +d.term) | Takefumi Yoshikawa | 737 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461202" abp="359">BBa_K1461202</A> | Composite | ecf11 (+RBS, +d.term) | Takefumi Yoshikawa | 764 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461203" abp="374">BBa_K1461203</A> | Composite | cr-ecf20 (+RBS, +d.term) | Takefumi Yoshikawa | 777 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461204" abp="389">BBa_K1461204</A> | Composite | cr-ecf11 (+RBS, +d.term) | Takefumi Yoshikawa | 804 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461205" abp="404">BBa_K1461205</A> | Composite | cr-GFP (+RBS, +d.term) | Takefumi Yoshikawa | 915 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461206" abp="419">BBa_K1461206</A> | Composite | anti ecf20 (+RBS, +d.term) | Takefumi Yoshikawa | 587 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461207" abp="434">BBa_K1461207</A> | Composite | anti ecf11 (+RBS, +d.term) | Takefumi Yoshikawa | 818 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461300" abp="959">BBa_K1461300</A> | Composite | RBS-GFP-4miR-142-3p bindng sites-d.term | Shunsuke Sumi | 1007 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461301" abp="974">BBa_K1461301</A> | Composite | RBS-GFP-4 miR-142-5p bindng sites-d.term | Shunsuke Sumi | 999 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461302" abp="989">BBa_K1461302</A> | Composite | RBS-GFP-miR 142 3p non-binding site-d.term | Shunsuke Sumi | 1001 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461303" abp="1004">BBa_K1461303</A> | Composite | RBS-GFP-miR 142 5p non-binding site-d.term | Shunsuke Sumi | 1007 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461304" abp="1019">BBa_K1461304</A> | Composite | RBS-GFP-2miR 142 3p bindng sites-2miR 142 5p bindng sites-d.term | Shunsuke Sumi | 1003 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461305" abp="1034">BBa_K1461305</A> | Composite | RBS-GFP-2miR 142 3p non-bindng sites-2miR 142 5p non-bindng sites-d.term | Shunsuke Sumi | 1003 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461208" abp="449">BBa_K1461208</A> | Generator | taRNA (arabinose-induced) (+d.term) | Takefumi Yoshikawa | 346 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461209" abp="464">BBa_K1461209</A> | Generator | arabinose-induced ecf20 generator | Takefumi Yoshikawa | 875 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461210" abp="479">BBa_K1461210</A> | Generator | constitutive ecf20 generator | Takefumi Yoshikawa | 780 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461211" abp="494">BBa_K1461211</A> | Generator | arabinose-induced ecf11 generator | Takefumi Yoshikawa | 902 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461212" abp="509">BBa_K1461212</A> | Generator | constitutive ecf11 generator | Takefumi Yoshikawa | 807 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461213" abp="524">BBa_K1461213</A> | Generator | arabinose-induced cr-ecf20 generator | Takefumi Yoshikawa | 915 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461214" abp="539">BBa_K1461214</A> | Generator | constitutive cr-ecf20 generator | Takefumi Yoshikawa | 820 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461215" abp="554">BBa_K1461215</A> | Generator | arabinose-induced cr-ecf11 generator | Takefumi Yoshikawa | 942 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461216" abp="569">BBa_K1461216</A> | Generator | constitutive cr-ecf11 generator | Takefumi Yoshikawa | 847 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461217" abp="584">BBa_K1461217</A> | Generator | arabinose-induced cr-GFP generator | Takefumi Yoshikawa | 1053 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461218" abp="599">BBa_K1461218</A> | Generator | constitutive cr-GFP generator | Takefumi Yoshikawa | 958 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461219" abp="614">BBa_K1461219</A> | Generator | ecf20-induced GFP generator | Takefumi Yoshikawa | 963 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461220" abp="629">BBa_K1461220</A> | Generator | ecf11-induced GFP generator | Takefumi Yoshikawa | 963 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461221" abp="644">BBa_K1461221</A> | Generator | ecf20 generator (positive feedback) | Takefumi Yoshikawa | 825 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461222" abp="659">BBa_K1461222</A> | Generator | ecf11 generator (positive feedback) | Takefumi Yoshikawa | 852 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461223" abp="674">BBa_K1461223</A> | Generator | anti ecf20 generator (pLac) | Takefumi Yoshikawa | 795 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461224" abp="689">BBa_K1461224</A> | Generator | anti ecf11 generator (pLac) | Takefumi Yoshikawa | 1026 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461225" abp="704">BBa_K1461225</A> | Measurement | ecf20 promoter measurement system | Takefumi Yoshikawa | 1846 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461226" abp="719">BBa_K1461226</A> | Measurement | ecf11 promoter measurement system | Takefumi Yoshikawa | 1873 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461231" abp="794">BBa_K1461231</A> | Measurement | arabinose-induced GFP and taRNA generator | Takefumi Yoshikawa | 1407 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461232" abp="809">BBa_K1461232</A> | Measurement | single counter | Takefumi Yoshikawa | 1312 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461233" abp="824">BBa_K1461233</A> | Measurement | ecf20 promoter mesurement system (positive feedback) | Takefumi Yoshikawa | 2679 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461234" abp="839">BBa_K1461234</A> | Measurement | ecf11 promoter mesurement system (positive feedback) | Takefumi Yoshikawa | 2733 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461235" abp="854">BBa_K1461235</A> | Measurement | resettable counter device using ecf20 (w/o crRBS, taRNA) | Takefumi Yoshikawa | 3482 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461236" abp="869">BBa_K1461236</A> | Measurement | resettable counter device using ecf11 (w/o crRBS, taRNA) | Takefumi Yoshikawa | 3767 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461237" abp="884">BBa_K1461237</A> | Measurement | sigma factors cross-talk assay 1 | Takefumi Yoshikawa | 1873 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461238" abp="899">BBa_K1461238</A> | Measurement | sigma factors cross-talk assay 2 | Takefumi Yoshikawa | 1846 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461227" abp="734">BBa_K1461227</A> | Project | resettable single counter system using ecf20 | Takefumi Yoshikawa | 2671 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461228" abp="749">BBa_K1461228</A> | Project | reset function using ecf20 | Takefumi Yoshikawa | 2576 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461229" abp="764">BBa_K1461229</A> | Project | resettable single counter system using ecf11 | Takefumi Yoshikawa | 2834 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461230" abp="779">BBa_K1461230</A> | Project | reset function using ecf11 | Takefumi Yoshikawa | 2929 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461239" abp="914">BBa_K1461239</A> | Project | σ-Re Counter (double, using ecf20) | Takefumi Yoshikawa | 3821 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461240" abp="929">BBa_K1461240</A> | Project | σ-Re Counter (double, using ecf11) | Takefumi Yoshikawa | 4106 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461241" abp="944">BBa_K1461241</A> | Project | σ-Re Counter (triple) | Takefumi Yoshikawa | 6615 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461104" abp="284">BBa_K1461104</A> | Regulatory | EpCAM promoter | Shunsuke Sumi | ||||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461105" abp="299">BBa_K1461105</A> | Regulatory | EpCAM promoter(mutant) | Shunsuke Sumi | ||||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461107" abp="314">BBa_K1461107</A> | Regulatory | EGP-2 | Shunsuke Sumi | ||||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461100" abp="224">BBa_K1461100</A> | RNA | miR-142-3p target binding site | ドリキャス❤ | 25 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461101" abp="239">BBa_K1461101</A> | RNA | miR-142-5p target bindng site | Shunsuke Sumi | 23 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461102" abp="254">BBa_K1461102</A> | RNA | miRNA-142 binding site control1 | Shunsuke Sumi | 23 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461103" abp="269">BBa_K1461103</A> | RNA | miRNA-142 binding site control2 | Shunsuke Sumi | 25 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461306" abp="1049">BBa_K1461306</A> | Translational_Unit | pCMV-RBS-GFP-4 miR 142 3p site-d.term | Shunsuke Sumi | 1595 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461307" abp="1064">BBa_K1461307</A> | Translational_Unit | pCMV-RBS-GFP-4 miR 142 5p site-d.term | Shunsuke Sumi | 1587 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461308" abp="1079">BBa_K1461308</A> | Translational_Unit | pCMV-RBS-GFP-4 miR 142 3p non-binding sites-d.term | Shunsuke Sumi | 1589 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461309" abp="1094">BBa_K1461309</A> | Translational_Unit | pCMV-RBS-GFP-4 miR 142 5p non-bindgin sites-d.term | Shunsuke Sumi | 1595 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461310" abp="1109">BBa_K1461310</A> | Translational_Unit | pCMV-RBS-GFP-2 miR 142 3p bindgin sites-2 miR 142 5p binding site-d.term | Shunsuke Sumi | 1591 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461311" abp="1124">BBa_K1461311</A> | Translational_Unit | pCMV-RBS-GFP-2 miR 142 3p non-bindgin sites-2 miR 142 5p non-binding site-d.term | Shunsuke Sumi | 1591 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461312" abp="1139">BBa_K1461312</A> | Translational_Unit | pEpCAM-RBS-GFP-4 miR 142 3p site-d.term | Shunsuke Sumi | 738 | |||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461313" abp="1154">BBa_K1461313</A> | Translational_Unit | pEpCAM-RBS-GFP-4 miR 142 5p site-d.term | Shunsuke Sumi | ||||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461314" abp="1169">BBa_K1461314</A> | Translational_Unit | pEpCAM-RBS-GFP-4 miR 142 3p non-bindng site-d.term | Shunsuke Sumi | ||||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461315" abp="1184">BBa_K1461315</A> | Translational_Unit | pEpCAM-RBS-GFP-4 miR 142 5p non-bindng site-d.term | Shunsuke Sumi | ||||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461316" abp="1199">BBa_K1461316</A> | Translational_Unit | pEpCAM-RBS-GFP-2 miR 142 3p bindgin sites-2 miR 142 5p binding site-d.term | Shunsuke Sumi | ||||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461317" abp="1214">BBa_K1461317</A> | Translational_Unit | pEpCAM-RBS-GFP-2 miR 142 3p non-bindgin sites-2 miR 142 5p non-binding site-d.term | Shunsuke Sumi | ||||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461318" abp="1229">BBa_K1461318</A> | Translational_Unit | pEpCAM(mutant)-RBS-GFP-4 miR 142 3p site-d.term | Shunsuke Sumi | ||||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461319" abp="1244">BBa_K1461319</A> | Translational_Unit | pEpCAM(mutant)-RBS-GFP-4 miR 142 5p site-d.term | Shunsuke Sumi | ||||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461320" abp="1259">BBa_K1461320</A> | Translational_Unit | pEpCAM(mutant)-RBS-GFP-4 miR 142 3p non-bindng site-d.term | Shunsuke Sumi | ||||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461321" abp="1274">BBa_K1461321</A> | Translational_Unit | pEpCAM(mutant)-RBS-GFP-4 miR 142 5p non-bindng site-d.term | Shunsuke Sumi | ||||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461322" abp="1289">BBa_K1461322</A> | Translational_Unit | pEpCAM(mutant)-RBS-GFP-2 miR 142 3p bindgin sites-2 miR 142 5p binding site-d.term | Shunsuke Sumi | ||||
| <A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461323" abp="1304">BBa_K1461323</A> | Translational_Unit | pEpCAM(mutant)-RBS-GFP-2 miR 142 3p non-bindgin sites-2 miR 142 5p non-binding site-d.term | Shunsuke Sumi |
7,14,2014
Lab member
Yoshikawa,Nakashima
Contents
Making Plate Culture
Ampicillin *10
Chloramphenicol*6
7,15,2014
Lab member
Yoshikawa,Nakashima
Contents
Making DW 100mL
TF
4-17F(A-1), 4-4E(A-2), 3-19O(A-3), 3-4G(A-4), 4-1N(A-6), 3-3F(A-8), 4-13L(B-10)
7,16,2014
Lab member
Yoshikawa,Nakashima
Contents
A-2:No colony
TF
A-2(recovery)
preculture
A-1, A-3, A-4, A-6, A-8, B-10,Escherichia coli JM109(for competent cell)
7,17,2014
Lab member
Yoshikawa,Nakashima
Contents
A-2:There are something like colonies.
Making glycerol stock/miniprep
A-1, A-3, A-4, A-6, A-8
All samples:consentration is low.
B-10:disposed
making reagent
LB: 500mL
50mM CaCl2: 400mL
50mM CaCl2/ 20% glycerol: 200mL
preculture
A-1 #1
A-2 #1 #2
A-3 #1
A-4 #1
A-6 #1
A-8 #1
B-10 #1 #2
E. coli JM109（for competent cell）
- colony number
7,18,2014
Lab member
Yoshikawa,Nakashima
Contents
miniprep
A-1,A-3,A-6:from culture 2.5ml OK
A-4,A-8:from culture 1.5ml OK
A-2#1:made glycerol stock. Low consentration.
B-10#1:Low consentration.
A-2#2,B-10#2:did not grow
Making competent cell
100uL*120
making reagent
（2000×）Ampicillin 3ｍL （100mg/mL）
7,22,2014
Lab member
Yoshikawa,Nakashima
Contents
Cut check
A-4 (81ng/μL):ES Cut,XP cut.Checked in 1.5h, 2h, 2.5h, 3h:OK
Left:1kbp Ladder/A-4(Control)/ES 1.5h/ES 2h/ES 2.5h/ES 3h
Right:1kbp Ladder/A-4(Control)/XP 1.5h/XP 2h/XP 2.5h/XP 3h
<img src="
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TF
We measured cfu of competent cell we made.
We used Efficiency Kit （RFP Construct on pSB1C3）in distribution Kit.
TF
50pg, 20pg, 10pg, 5pg
competent cell:25uL
preculture
B-10 #3, A-4
7,23,2014
Lab member
Yoshikawa,Nakashima
Contents
plate check:Did not grow.
miniprep
A-4, B-10 #3(made glycerol stock):OK
Making plate
200ml,CP*10
7,28,2014
Lab member
Nakashima
Contents
digestion,gel extraction
A-6 SP, B-10 XP：disposed
100bp Ladder, A-6 SP, B-10 XP, NC
<img src="
"/>
(wrote at 0731
A-6
band near 2kbp:SP cut or single cut
band near 1.5kbp:Plasmid)
colony PCR
A-6 #1, B-10 #1
1kbp Ladder, A-6, B-10, NC
<img src="
"/>
A-6:OK,B-10:wrong
Measuring cfu
competent cell:50uL
7,29,2014
Lab member
Yoshikawa,Nakashima
Contents
Plate check:All Plates did not grow.
TF
B-10 by electroporation.
Measuring cfu
A-1:13,65,130pg
Electrophoresis check
1kbp Ladder, A-6(Plasmid), NC
<img src="
"/>
There is band in 1500kbp,so 1500kbp in 0728 may be rest of cutting.
7,30,2014
Lab member
Yoshikawa,Nakashima
Contents
colony check
cfu:Did not grow
B-10:One colony
Cut check
We checked whether A-6 band in 0728 was rest of cutting.
colony PCR
B-10(from Plate made in 0729)
1kbp Ladder, 1.5h, 2h, 2.5h, B-10(colony PCR), NC
<img src="
"/>
band near 800bp:RFP between Spe1 site and Pst1 site of A-1.
band near 2kbp:Backbone cut by SP cut
band near 3kbp:rest of cutting
1.5h:incompletely cut
2h,2.5h:OK
We decided 2h for digestion.
B-10:OK
preculture
B-10
7,31,2014
Lab member
Yoshikawa,Nakashima
Contents
miniprep
B-10 #1（from 140729 Plate）(made glycerol stock)
digestion,gel extraction
A-6 SP, B-10 XP
1kbp Ladder, A-6, B-10
<img src="
"/>
B-10:incomplete cut
others:OK
ligation
C-7 (A-6 SP + B-10 XP)
8,1,2014
Lab member
Yoshikawa,Nakashima
Contents
TF
C-7（Chemical & EP）
ligation Check
PCR reaction primed with Universal Primer, using ligation products as template.
<img src="
"/>
OK!
8,4,2014
Lab member
Yoshikawa,Nakashima
Contents
Plate check
No colony
digestion
A-6 SP, B-10 XP
Electrophoresis
1kbp Ladder, A-6, B-10
<img src="
"/>
Incomplete cut.Disposed.
preculture
B-10
8,5,2014
Lab member
Yoshikawa,Nakashima
Contents
miniprep
B-10 →Sample Lost! Bye bye, GFP!
Cut check
SP(A-1), XP(A-1), EX(A-4), ES(A-4)
2h, 2.5h, 3h
Elrctrophoresis
1kbp Ladder, A-1, A-1 SP(2h, 2.5h, 3h), A-1 XP(2h,2.5h, 3h), A-4,
A-4 EX(2h, 2.5h 3h), A-4 ES(2h, 2.5h, 3h), None, 1kbp Ladder
<img src=""/>
Incomplete cut.
preculture
A-1, A-4, B-10
8,6,2014
Lab member
Yoshikawa
Contents
miniprep
A-1, A-4, B-10
digestion, gel extraction
A-6 SP, B-10 XP
1kbp Ladder, A-6, B-10
<img src="
"/>
A-6:OK
B-10:Incomplete cut
<img src="
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We chenged restriction enzyme.
B-10 XP
<img src="
"/>
OK!
<img src="
"/>
making gel
200ml
ligation
C-7（A-6 SP + B-10 XP）
TF
4-11L, C-7
8，7,2014
Lab member
Yoshikawa,Nakashima,Tara,Itoh,Tsukada
Contents
colony PCR
C-7
<img src="
"/>
<img src="
"/>
All bands are self ligation of backbone.
digestion, gel extraction
A-6 SP, B-10 XP
1kbp Ladder, B-10, A-6
<img src="
"/>
ligation
C-7 (A-6 SP + B-10 XP)
Cut check
(O/N)
（enzyme-buffer)
S-B, S-H*, S-M*, P*-FD, E-FD
- Takara's product
- Takara's product
8,8,2014
Lab member
Nakashima,Nakamura,Yamanaka,Yoshikawa(Fresh),Yoshikawa
Contents
Cut check sample Electrophoresis
1kbp Ladder, S-B, S-H*, S-M*, NC, E-FD, P*-FD
<img src="
"/>
FD buffer is appropriate for SP cut.
digestion, gel extraction
A-6 SP, B-10 XP
<img src="
"/>
<img src="
"/>
OK!
ligation
C-7 (A-6 SP + B-10 XP)
TF
C-7,4-11L(culture in test tube)
overlap extension PCR →PCR clean up
1+2
anealing 57℃,extension 10 sec, 30 cycle.
100bp Ladder, Pecf11, Pecf20, crRBS, taRNA
<img src="
"/>
OK!(low concentration)
making gel
200ml
8,9,2014
Lab member
Yoshikawa
Contents
Plate and test tube check
4-11L:OK!mixed with glycerol, and conserved in -80℃.
C-7:OK! conserved in refrigerator.
8,11,2014
Lab member
Yoshikawa,Nakashima,Yamanaka,Nakamura
Contents
colony PCR
C-7 #1~11
- 1~4, NC
<img src="
"/>
- 5~11,NC
<img src="
"/>
- 10 is OK!
overlap extension PCR →PCR clean up
Pecf11, Pecf20, crRBS, taRNA
(1+2)+3,PCR product
1kbp Ladder, Pecf11(1+2+3), Pecf20(1+2+3), crRBS(1+2+3)
taRNA(1+2+3), Pecf11(all), Pecf20(all), crRBS(all), taRNA(all)
<img src=""/>
Making Plate
Ampicillin*20
preculture
A-6, B-10, C-7 #10
8,12,2014
Lab member
Nakashima,Nakamura,Yamanaka,Yoshikawa(Fresh),Yoshikawa
Contents
miniprep
A-6, B-10, C-6 #10 (made glycerol stock)
digestion →gel extraction
A-8 EX, C-6 ES, pSB1A2(A-1) XP *2 sample, A-9 linear XP
A-10 linear XP, A-7 linear XP, B-1 linear XP
1kbp Ladder, A-1 XP, A-1 XP, A-8 EX, C-6 ES
<img src="
"/>
C-6:wrong
A-1:OK!
colony PCR
4-11L
<img src="
"/>
OK!
ligation →TF
A-9, A-10, A-7, B-1
Making reagent
LB 800ml
8,13,2014
Lab member
Yoshikawa,Nakashima,Tara,Nakamura,Takemura,Tsukada
Contents
miniprep
4-11L（made glycerol stock）
digestion→ gel extrction
A-8 EX, C-6 ES, A-6 SP, 4-11L XP
1kbp Ladder, A-6 SP, A-8 EX, C-6 ES, 4-11L XP
<img src="
"/>
<img src="
"/>
C-6:Incomplete cut
Backbone of 4-11L said to be pSB1A2, but ,actually,may be pSB1AK3.
ligation→TF
D-7 (4-11L XP + A-6 SP), D-7(C-6 ES + A-8 EX)
colony PCR
A-7 #1~2, A-9 #1~4, A-10 #1~4, B-1 #1~2 ,100bp Ladder
<img src="
"/>
A-7 #1, A-10 #4：OK!
preculture
A-8, C-6, A-7 #1, A-10 #4
8,14,2014
Lab member
Yoshikawa,Nakashima,Tara,Takemura,Nakamura
Contents
miniprep
A-7 #1（made glycerol stock）, A-10 #4（made glycerol stock）, A-8, C-6
overlap extension PCR
Pecf 11, Pecf 20, crRBS, taRNA
(35 cycles, annealing in 59℃)
digestion → gel extranction
A-7 SP, 4-11L XP, pSB1A2 (B-10), A-7 linear XP
A-9 linear XP, A-10 linear XP, B-1 linear XP
1kbp Ladder, A-7 SP, 4-11L XP
<img src="
"/>
<img src="
"/>
OK!
A-10 XP, 100bp Ladder
<img src="
"/>
OK!(low concentration)
100bp Ladder, pSB1A2(B-10), pSB1A2(B-10), A-7, A-9, B-1
<img src="
"/>
<img src="
"/>
Incomplete cut of pSB1A2 is weigh on our mind.
gel making
2% 25mL
1% 200mL
colony PCR
D-7(Ampicillin), D-7(Chloramphenicol)
1kbp Ladder, D-7(Amp) #1~4, D-7(CP) #1~4
<img src="
"/>
ligation → TF
D-6 (A-7 SP + 4-11L XP), A-7 (linear XP + pSB1A2 XP)
A-9, A-10, B-1 (linear XP + pSB1A2 XP)
8,15,2014
Lab member
Yoshikawa,Nakashima,Tara,Takemura,Nakamura
Contents
miniprep
D-7 #3 (made glycerol stock)
digestion → gel extraction
A-10 SP, D-7 XP
<img src="
"/>
<img src="
"/>
colony PCR
A-7 #1~4, A-9 #1~4, A-10 #1~4, B-1 #1~4, D-6 #1~4
<img src="
"/>
A-7 #3,4, A-10 #1,2,4, B-1 #1,2,4：OK!
D-6
<img src="
"/>
OK!
ligation → TF
E-18 (A-10 SP + D-7 XP)
preculture
A-7 #3,4, A-10 #1,2,4, B-1 #1,2,4, D-6 #1
8,16,2014
Lab member
Yoshikawa
Contents
miniprep
A-7 #3,4, A-10 #1,2,4, B-1 #1,2,4, D-6 #1
(made glycerol stock)
8,18,2014
Lab member
Yoshikawa,Hiura
Contents
digestion → gel extraction
A-1 SP, A-2 SP, A-3 SP, D-6 XP *2, A-7 #3,4, B-10
<img src="
"/>
sequence
A-7, A-7 #3,4, A-10, A-10 #1,2,4, B-1 #1,2,4
C-6 F, C-6 R, D-7 F, D-7 R, D-6 F, D-6 R
colony PCR
E-18 #1~3
<img src="
"/>
OK!
ligation → TF
E-14 (A-3 SP + D-6 XP)
E-15 (A-1 SP + D-6 XP)
E-16 (A-2 SP + D-6 XP)
preculture
E-18 #1
8,19,2014
Lab member
Yoshikawa,Nakamura,Yamanaka,Yoshikawa(Fresh)
Contents
miniprep
E-18 (made glycerol stock)
digestion
A-8 XP, B-1 #1,2,4 SP
→Today, sequences of these dna proved to be wrong, so we disposed them.
making gel
2% gel:25ml
colony PCR
C-5, E-14, E-15,E-16
sequence
A-7:RBS
A-7 #3:none
A-7 #4:none
A-10:OK
B-1: all none
8,20,2014
Lab member
Yoshikawa,Nakamura,Tsukada,Yamanaka,Itoh
Contents
PCR
sigma11, sigma20, anti11, anti20
TF
1-21P
8,21,2014
Lab member
Yoshikawa,Nakashima
Contents
digestion → gel extraction
pSB1C3(A-4), A-6, B-3(We did not extract.), A-6'(NEB buffer)
<img src="
"/>
colony PCR
A-2, A-3, A-4, 1-21P, NC
<img src="
"/>
ligation → TF
B-3, C-2, C-2'
preculture
1-21P
8,22,2014
Lab member
Yoshikawa,Nakamura,Nakashima
Contents
miniprep
1-21P(made glycerol stock)
colony PCR
B-3, C-2, C-2
<img src="
"/>
digestion → gel extraction
1-21P SP, D-7 XP, C-6 E, C-6 P, C-6(NC)
<img src="
"/>
<img src="
"/>
ligation → TF
K-1(1-21P SP + D-7 XP)
preculture
B-3 #1,4, D-7, C-2 #1,3, C-2' #3,4
8,23,2014
Lab member
Yoshikawa
Contents
miniprep
D-7, B-3 #1,4, C-2 #1,3, C-2' #3,4
8,25,2014
Lab member
Nakashima,Nakamura,Yamanaka,Hiura
Contents
digestion → gel extraction
A-8 EX *2, B-3 #1 ES, B-3 #4 ES, C-2 #1 ES, C-2 #3 ES
<img src="
"/>
<img src="
"/>
Plate making
Ampicillin*10, Chloramphenicol*20
colony PCR
K-1
<img src="
"/>
- 1,4:OK!
sequence
B-3 #1,4, C-2 #1,3, C-2' #3,4, E-18, A-2, A-3
ligation → TF
D-3 1 (A-8 EX + C-2 #1 ES), D-3 2(A-8 EX + C-2 #3 ES)
C-10 1(A-8 EX + B-3 #1 ES), C-10 2(A-8 EX + B-3 #4 ES)
preculture
A-2, A-8, K-1 #1, C-2 #1,3, B-3 #1
8,26,2014
Lab member
Yoshikawa,Nakashima,Yamanaka,Takemura,Tsukada
Contents
miniprep
K-1(made glycerol stock), A-2, A-8, B-3 #1, C-2 #1, C-2 #3
colony PCR
D-3 (1), D-3 (2), C-10 (1), C-10 (2)
<img src="
"/>
OK!(C-10(1)#4 may be a little shorter.)
digestion → gel extraction
K-2 linear EP, K-3 linear EP, K-4 limear EP, K-5 linear EP, pSB1C3 (A-4) EP
<img src="
"/>
We forgot to take the photo before we sliced the band.
ligation → TF
K-2, K-3, K-4, K-5
preculture
D-3 #1, C-10 #1
sequence
A-2:E-X-S-S-Pconst(weak)-S-P wrong
sequence
A-3:OK
B-3 #1:OK
B-3 #4:OK
C-2 #1:OK
C-2 #3:OK
C-2' #3:point mutation *2
C-2' #4:OK
E-18:OK
8,27,2014
Lab member
Yoshikawa,Nakashima,Yoshikawa(fresh),Itoh,Tara,Nakamura
Contents
miniprep
D-3, C-10(made glycerol stock)
digestion → gel extraction
A-1 SP, A-3 SP, A-10 SP, D-3 XP *2
<img src="
"/>
<img src="
"/>
D-3 is a little strange.Contamination?
colony PCR
K-2~5 #1~4
- 100bp Ladder
<img src="
"/>
500bp band:self ligation of backbone(A-4 200bop)
300bp band:If this band is blank vector, this band(EP cut) must be shorter than 300bp.
So, this band is OK!.
→K-2 #4, K-3 #1, K-4 #1,2, K-5 #2,3,4:OK!
ligation → TF
E-5(A-3 SP + D-3 XP), E-6(A-1 SP + D-3 XP), E-20(A-10 SP + D-3 XP)
preculture
E-15
D-3, C-10(Recovery)
K-2 #4, K-3 #1, K-5 #2
Remarks
<img src="
"/>
We observed that pCMV expressed in Escherichia.coli.
Left:pCMV-GFP
Right:pConst(strong)-GFP
We may be able to carry out the characterization of pCMV and meet the gold medal requirement(parts implovement).
8,28,2014
Lab member
Yoshikawa,Nakashima,Itoh,Tara,Tsukada,Yamanaka
Contents
miniprep
K-2~5, E-23(made glycerol stock)
C-10, D-3
digestion →gel extraction
K-2 EX, K-3 EX, K-4 EX, K-5 EX, E-23 EP, C-6 ES *2, pSB1C3(A-4) EP, pSB1C3(A-4) XP
<img src="
"/>
<img src="
"/>
→pSB1C3 XP:keep in freezer
overlap extension PCR
A-7, A-9, B-1, B-2, D-8, D-9
check & colony PCR
check:E-5, E-6
colony PCR:(E-5 #1~4, E-6 #1~4, NC), A-7, A-9, D-8, B-1, B-2, D-9
<img src="
"/>
A-7, A-9, B-1, D-8:OK!
B-2, D-9:Needs retry in higher concentration.
E-5 #2~4, E-6:OK!
ligation → TF
E-23'(E-23 EP + pSB1C3 EP), L-1~4(C-6 ES + K-2~5 EX)
8,29,2014
Lab member
Yoshikawa,Nakashima,Nakamura,Tara,Tsukada,Yamanaka
Contents
miniprep
E-5(made glycerol stock)
A-4, C-6
E-6:No colony
digestion →gel extraction
A-7 linear XP, A-9 linear XP, A-10 SP, B-1 linear XP, D-3 XP, D-8 linear XP, E-5 SP, E-18 XP
<img src="
"/>
<img src="
"/>
PCR
B-2, D-9(retry)
check & colony PCR
E-23' #1~4, B-2, D-9
<img src="
"/>
L-1~4
<img src="
"/>
E-23 #1, L-1 #2,4, L-2 #4, L-3 #1~3, L-4 #2,4:OK!
PCR:failed
sequence order
E-23, K-1, K-2~5
ligation → TF
A-7, A-9, B-1, D-8( linear XP + pSB1C3 XP)
F-2 (E-18 XP + E-5 SP), E-20(A-10 SP + D-3 XP)
preculture
A-10, E-6, E-18, E-23', K-3, L-1~4
8,30,2014
Lab member
Yoshikawa
Contents
miniprep
A-10, E-18, K-3
L-1~4, E-23', E-6, E-18(made glycerol stock)
PCR
B-2, D-9
9,1,2014
Lab member
Nakashima,Itoh,Tara,Tsukada,Takemura
Contents
PCR clean up
B-2, D-9
<img src="
"/>
failed
digestion → gel extraction
L-4 ES, E-6 ES, E-18 EX, L-1~3 ES, K-2~5 EX
<img src="
"/>
<img src="
"/>
Making plate
CP *30
colony PCR
A-7 #1~4, A-9 #1
<img src="
"/>
OK!
D-8 #1~4, E-20 #1~4, F-2 #1~4 (100bp Ladder)
<img src="
"/>
D-8 #1~4, E-20 #1~4,F-2#1,4:OK!
B-1 #1~4
<img src="
"/>
OK!
Ligation
G-5 (E-6 ES + E-18 EX), M-1~4(K-2~5 EX + L-1~4 ES)
preculture
A-7 #1~4, A-9 #1, B-1 #1~4, D-8 #1~4, F-2, E-20
9,2,2014
Lab member
Yoshikawa,Nakashima,Hiura,Tara,Tsukada,Takemura
Contents
miniprep
A-7 #1~4, A-9, B-1 #1~4, D-8 #1~4, F-2, E-2(made glycerol stock)
digestion → gel extraction
A-4 SP, A-7 SP #1, A-7 SP #2, A-9 SP
<img src="
"/>
<img src="
"/>
A-8XP
<img src="
"/>
<img src="
"/>
B-1 #1, B-1 #2, B-3 XP, B-10 XP, D-7 XP, D-8 XP, E-20 XP, F-2 SP
<img src="
"/>
<img src="
"/>
D-8:???
sequence
A-7 #1~4, B-1 #1~4
D-8 #1~4
A-9, A-4, E-5, E-6, E-20
PCR
B-2, D-9
check & colony PCR
M-1~3 #1~4
<img src="
"/>
OK!
M-4 #1~4, B-2, D-9, PC(VF2→B-10←VR )
NC, G-5
<img src="
"/>
M-4 #1~4,PC(VF2→B-10←VR ):OK!
(M-4 #2~4 is a little longer?)
ligation → TF
C-4(A-7 SP + B-3 XP), C-5(A-7 SP + B-10 XP), E-17(A-9 SP + D-7 XP)
D-1(B-1 SP + D-8 XP), E-21(A-4 SP + D-8 XP), J-4 (F-2 SP + E-20 XP)
preculture
M-1~4 #1, G-5 #1, E-20
We got a result!!
simpler version of assay 1.
Left:Pσ11→GFP generator
Right:Pconst(strong)→σ11 generator & Pσ11→GFP generator
<img src="
"/>
9,3,2014
Lab member
Yoshikawa,Nakashima,Hiura,Tara,Yamanaka,Takemura
Contents
M-1~4, G-5(made glycerol stock of M-3,4)
sequence
A-4: OK
A-7: all OK
A-9: OK
B-1:all OK
D-8#1: NG (2 skip)
#2; OK
#3: NG (1 mut.)
#4; NG (2 mut. 2 skip)
E-5: OK
E-6: OK
E-20:OK
digestion → gel extraction
A-7 #1, 2 SP, C-9 XP, C-10 XP, K-2~4 EX, M-1~4 ES
<img src="
"/>
<img src="
"/>
colony PCR
C-4 a, C-5 a, D-1 a, E-17
<img src="
"/>
C-4 a#2,3, C-5 a, D-1 a, E-17:OK!
E-21 b, J-4
<img src="
"/>
J-4:wrong
E-21:a little shorter
PCR
B-2, D-9, B-7, B-2(Taq), D-9(Taq)
Taq:positive control
anealing temperature:51℃～61℃(gradient)
ligation → TF
D-5 (A-7 SP + C-10 XP), D-6 (A-7 SP + C-9 XP), N-1~4 (M-1~4 ES + K-2~5 EX)
N-5 (M-1 ES K-3 EX), N-6 (M-3 ES + K-5 EX)
9,4,2014
Lab member
Yoshikawa,Nakashima,Nakamura,Tara,Yamanaka,Tsukada
Contents
miniprep
E-20, D-1(made glycerol stock)
digestion → gel extraction
pSB1C3 (A-4) XS, pSB1C3 (A-4) XP, A-3 SP, D-1 XP
<img src="
"/>
<img src="
"/>
PCR check & clean up
D-9 #1,3,5,7,9
<img src="
"/>
There are bands in #5,7,9
B-2 1,3,5,7,9,11, B-7.
<img src="
"/>
There are bands in B-2 #5,7, B-7.
B-2 4,6, D-9 10,11,12
<img src="
"/>
OK!
We decided to clean up B-2 #5, D-9 #11, B-7.
Making Plate
CP *30
digestion
B-2 linear SP, D-9 linear XP, B-7 linear XP
colony PCR
D-5, D-6, N-1, N-2 #1~4
<img src="
"/>
D-5, D-6, N-1, N-2 #1,2:OK!
N-3~6 #1~4
<img src="
"/>
N-3,N-4#1,3,4,N-5#1,4,N-6:OK!
ligation → TF
E-1 (A-3 SP + D-1 XP), B-2 (B-2 linear XP + pSB1C3 XP), D-9 (D-9 linear XP + pSB1C3 XP)
B-7 (B-7 linear XP + pSB1C3 XP), Emp. (pSB1C3 XS)
miniprep
M-1, M-2, C-4, C-5, E-17, E-21, J-4 (made glycerol stock)
K-2, K-5, C-9
preculture
F-2, D-5, D-6, N-1~6
9,5,2014
Lab member
Yoshikawa,Nakashima,Nakamura,Takemura,Yamanaka
Contents
miniprep
F-2
N-1~6, D-5, D-6
→N-1, 2, 4, 5. 6 :failed
digestion → gel extraction
A-1 SP, A-3 SP, D-5 XP, D-6 SP, E-20 XP, K-4 EX, N-3 ES, F-2 SP
J-4 E(check)
<img src="
"/>
<img src="
"/>
ligation → TF
E-11 (A-3 SP + D-5 XP), E-12 (A-1 SP + D-5 XP), E-14 (A-3 SP + D-6 SP)
E-15 (A-1 SP + D-6 XP), O-3 (K-4 EX + N-3 ES)
PCR
B-2, D-9, B-7
colony PCR
B-2, B-7, D-9, E-1 #1~4
<img src="
"/>
B-7 #2,3, D-9 #1, E-1 #3,4:OK!
Z-1 #2~4
<img src="
"/>
preculture
B-7 #2,3, D-9 #1, E-1 #3, Z-1 #2, K-3, N-1,2,4~6
9,6,2014
Lab member
<p>Yoshikawa,Nakashima
Contents
miniprep
B-7 #2,3, D-9 #1, E-1 #3, Z-1 #2 (made glycerol stock)
N-1,2,4~6, K-3
PCR
B-2, D-9, B-7
check & colony PCR
B-2, D-9, B-7, B-2#5~8, B-7 #2,3,5,6, D-9 #5,6.7,8
<img src="
"/>
9,8,2014
Lab member
Yoshikawa,Nakashima,Nakamura,Takemura,Yamanaka,Tara
Contents
digestion → gel extaction
B-2 linear XP (did not extracted)
N-1,2,4~6 ES
<img src="
"/>
<img src="
"/>
A-4 SP, pSB1C3 (A-4) XP, A-6 SP, B-7 #2 XP, B-7 #3 XP, D-9 #1 XP, K-2,3,5 EX
<img src="
"/>
B-7 #2, D-9 #1:disposed(incomplete cut)
B-7 #3:disposed(a little longer)(This band proved to be correct.9/9 wrote)
<img src="
"/>
colony PCR
E-11,12,14,15 #1~4
<img src="
"/>
OK!
O-3 #1,2
<img src="
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- 2:OK!
PCR
B-2
<img src="
"/>
B-7,D-9
<img src="
"/>
The band of the longest DNA is OK!
ligation → TF
O-1 (N-1 ES + K-2 EX), O-2 (N-2 ES + K-3 EX), O-4 (N-4 ES + K-5 EX), O-5 (N-5 ES + K-3 EX)
O-6 (N-6 ES + K-5 EX), B-2 (B-2 linear XP (9/8 PCR) + pSB1C3 XP)
B-2' (B-2 linear XP + pSB1C3 XP), D-9, B-7
preculture
D-5, D-6, N-3, E-21, A-3, K-4
E-11 #1, E-12 #1, E-14 #1, E-15 #1, O-3 #2, B-7 #6, D-9 #5,6
9,9,2014
Lab member
Yoshikawa,Nakashima,Nakamura,Takemura,Yamanaka
Contents
miniprep
E-11,12,14,15, O-3, B-7 #6, D-9 #5,6 (made glycerol stock)
D-5, N-3, K-4, A-3
D-6:did not grow
D-5:diposed(doubt of cross contamination)
digestion → gel extraction
B-2 linear XP (did not extracted)
pSB1C3 (A-4) XP, A-6 SP, A-8 EX, B-7 #6 XP, D-9 linear XP, B-7 linear , E-1 EX, E-14 ES, E-15 ES, O-3 ES
<img src="
"/>
<img src="
"/>
colony PCR
B-2 #1~3, B-2' #1~4, B-7 #1~3, D-9 #1,2, O-1 #1~4
<img src="
"/>
B-2' #1, B-7 #1,2, O-1:OK!
O-2,4,5,6 #1~4
<img src="
"/>
O-2,3,4,6,O-5#2,3,4
sequence
B-7 #2: NG
B-7 #3: OK
D-9 #1: NG
E-1: OK
C-4: OK
C-5: OK
D-5: OK
D-6: OK
E-17: OK
E-21: OK
F-2: ?
J-4: OK
N-1: OK
N-2: M-2
N-3: OK
N-4: M-4
N-5: OK
N-6: OK
ligation → TF
D-9 (D-9 linear XP + pSB1C3 XP) *2, B-2 (B-2 linear XP + pSB1C3 XP)*2
P-3 (O-3 ES + A-8 EX), I-1 (E-14 ES + E-1 EX), I-2 (E-15 ES + E-1 EX)
preculture
F-2, D-5,6, D-9 #5,6, E-12, K-4
O-1,2,4,6 #1, O-5 #2, B-2' #1
9,10,2014
Lab member
Nakashima,Hiura,Takemura,Yamanaka,Yoshikawa(Fresh)
Contents
miniprep
O-1,5,6, N-2,4, F-2, B-2' #1 (made glycerol stock)
E-12, K-4, D-5
D-6:did not grow
digestion → gel extraction
A-6 SP, A-7 SP, A-8 EX, B-2' XP, B-7 XP
<img src="
"/>
<img src="
"/>
K-3 EX, K-5 EX, O-1 ES, N-2 ES, N-4 ES
<img src="
"/>
<img src="
"/>
O-5 ES, O-6 ES
<img src="
"/>
<img src="
"/>
colony PCR
I-1 #1~4
<img src="
"/>
B-2(140909) #1~8, D-9 #1~8
<img src="
"/>
B-2 #2, D-9 all, I-1 #2~4, I-2 #1~4:OK!
ligation → TF
O-2 (N-2 ES + K-3 EX), O-4 (N-4 ES + K-5 EX), P-1 (O-1 ES + A-8 EX), P-5 (O-5 ES + A-8 EX)
P-6 (O-6 ES + A-8 EX), C-1' (B-2' XP + A-6 SP), C-3' (B-2 ' XP + A-7 SP), C-8 (B-7 XP + A-6 SP)
preculture
D-9 #5,6, D-9(140909) #1~4, B-2 (140909) #2, I-1 #2, I-2 #1, D-6 #1
9,11,2014
Lab member
Yoshikawa,Nakashima,Nakamura,Hiura,Itoh,Tsukada
Contents
miniprep
D-9 #1~4, B-2 #2, I-1, I-2, D-6 (made glycerol stock)
digestion → gel extraction
A-1 EP, pSB1C3(A-3) EP *2, A-4 SP, A-6 SP, A-7 SP, A-8 EX, A-10 EX, B-2 #2 XP, E-6 EP, E-12 EP
<img src="
"/>
<img src="
"/>
E-15 EP, E-18 EP, E-20 EP, D-9 #1 XP, O-3 ES
<img src="
"/>
<img src="
"/>
sequence
B-2' #1, B-2 #2, F-2, E-11, E-12, E-14, E-15, O-1, N-2, O-3, N-4, O-5, O-6, D-9 #1~4
Plate Making
CP *30
colony PCR
C-1', C-3', C-8, O-2, O-4 #1~3
<img src="
"/>
C-1', C-3', C-8, O-2, O-4 #1,2:OK!
P-1, P-5, P-6 #1~3
<img src="
"/>
→P-1 #1~3, P-5 #2, P-6 #1~3:OK!
ligation → TF
C-1 (A-6 SP + B-2 #2 XP), C-3 (A-7 SP + B-2 #2 XP)
E-22a (A-4 SP + D-9 #1 XP), E-22b (A-4 SP + D-9 #2 XP), P-3 (O-3 ES + A-8 EX)
A-10 CP, A-1 CP, E-6 CP, E-12 CP, E-20 CP, E-15 CP (replace backbone to pSB1C3)
preculture
C-1' #1, C-3' #1, C-8 #1, O-2 #1, O-4 #1, P-1 #1, P-5 #2, P-6 #1, E-12,14,15,18, A-1, O-1,3,5
9,12,2014
Lab member
Yoshikawa,Nakashima,Hiura,Itoh,Tsukada,Tara
Contents
miniprep
C-1', C-3', O-2, O-4, P-1, P-5, P-6 (made glycerol stock)
E-12, E-14, E-15, O-1, O-3, O-5, E-18, A-1
digestion → gel extraction
A-6 SP, A-8 EX *2, B-2 XP, C-1' ES, C-3' ES, C-8 ES, K-6 SP, O-2 ES, O-3 ES, O-4 ES
<img src="
"/>
<img src="
"/>
C-1,3:did not extracted
P-1 XP, P-5 XP, P-6 XP
<img src="
"/>
<img src="
"/>
colony PCR
A-1 CP, A-10 CP, C-3, E-6 CP #1~3
<img src="
"/>
E-12 CP, E-15 CP, E-20 CP, E-22a, E-22b
<img src="
"/>
except E-20#1,2:OK!
sequence
B-2' #1: NG
B-2 #2: OK
D-9 #1: NG
#2: NG
#3: NG
#4: OK
O-1: OK
O-3: OK
O-5: OK
O-6: OK
N-2: OK
N-4: OK
E-11: Ok
E-12: OK
E-14: OK
E-15: OK
F-2: OK
ligation → TF
C-1 (A-6 SP + B-2 XP), P-2~4 (O-2~4 ES + A-8 EX), Q-1,5,6 (P-1,5,6 XP + K-6 SP)
ligation
P-3 (O-3 ES + A-8 EX)
preculture
A-4, A-6, A-8, C-3 #1, A-1 CP #1, E-12 CP #1, E-15 CP #1, E-20 CP #3, E-6 CP #1
9,13,2014
Lab member
Yoshikawa
Contents
miniprep
A-4, A-6, A-8
C-3, E-6 CP, E-12 CP, E-15 CP, E-20 CP, A-1 CP(made glycerol stock)
9,15,2014
Lab member
Yoshikawa,Nakashima,Takemura,Nakamura,Yamanaka,Tara
Contents
digestion → gel extraction
A-8 EX, C-3 ES
<img src="
"/>
<img src="
"/>
colony PCR
C-1 (σ20F, VR)
<img src="
"/>
OK!
ligation → TF
D-4 (C-3 ES + A-8 EX) *2 (new and old competent cell)
preculture
C-1 #1
9,16,2014
Lab member
Yoshikawa,Nakashima,Hiura,Itoh,Nakamura,Yamanaka
Contents
miniprep
C-1 (made glycerol stock)
colony PCR
D-4, P-2~4
<img src="
"/>
A-10
<img src="
"/>
Q-1,5,6:confirmed by fluorescence
digestion → gel extraction
A-4 SP, A-8 EX, C-1 ES, D-9 XP, E-18 EP, pSB1C3 (A-4) EP
<img src="
"/>
ligation → TF
D-2 (A-8 EX + C-1 ES), E-18 CP (E-18 EP + pSB1C3 EP) *2, E-22 (A-4 SP + D-9 XP)
preculture
D-4, P-2~4, A-10 CP, Q-1,5,6
assay
<img src="
"/>
PC (E-23': Pconst (strong)-GFP-d.term)
Experiment (K-1: pCMV-GFP-d.term)
NC (Z-1: pSB1C3)
absorbance:600nm and 395nm
Absorbance of 395nm proved not to be able to measure.
We need fluorospectro-photometer.
9,17,2014
Lab member
Yoshikawa,Nakashima,Hiura,Takemura,Yoshikawa(Fresh),Yamanaka
Contents
miniprep
D-4, P-2~4, Q-1,5,6, A-10 CP (made glycerol stock)
digestion → gel extraction
A-1 CP SP, A-3 SP, D-4 XP, K-6 SP, P-2 XP, P-3 XP, P-4 XP
<img src="
"/>
<img src="
"/>
colony PCR
D-2, E-18 CP, E-22
<img src="
"/>
ligation → TF
E-8 *2 (A-3 SP + D-4 XP), E-9 (A-1 CP SP + D-4 XP), Q-2~4 (K-6 SP + P-2~4 XP)
assay
Photo of plate(n=4)
<img src="
"/>
<img src="
"/>
<img src="
"/>
<img src="
"/>
<img src="
"/>
<img src="
"/>
<img src="
"/>
<img src="
"/>
9,18,2014
Lab member
Yoshikawa,Nakashima,Hiura,Tsukada,Nakamura
Contents
miniprep
D-2, E-18 CP, E-22
(made glycerol stock)
digestion → gel extraction
A-1 CP SP, A-3 SP, A-9 SP, D-2 XP, E-22 SP, G-5 XP
<img src="
"/>
<img src="
"/>
colony PCR
E-8, E-9 #1~4
<img src="
"/>
Q-2~4 :confirmed by fluorescent
ligation → TF
E-2 (A-3 SP + D-2 XP)
E-3 (A-1CP SP + D-2 XP)
E-19 (A-9 SP + D-2 XP)
H-5 (E-22 SP + G-5 XP)
preculture
E-8,9, Q-2~4
9,19,2014
Lab member
Yoshikawa,Nakashima,Hiura,Tara,Yamanaka,Nakamura
Contents
miniprep
E-8,9, Q-2~4
digestion → gel extraction
A-9 SP, D-2 XP, E-22 XP, F-2 SP, G-5 SP
<img src="
"/>
<img src="
"/>
colony PCR
E-2, E-19 #1~4
E-3, H-5 :No colony
<img src="
"/>
ligation → TF
H-5 (G-5 SP + E-22 XP)
H-4 (F-2 SP + E-22 XP)
preculture
E-2 E#1, E-19 #1
9,20,2014
Lab member
Yoshikawa
Contents
miniprep
E-2, E-19
9,22,2014
Lab member
Yoshikawa,Nakashima,Takemura,Nakamura,Tara,Yamanaka
Contents
digestion → gel extraction
A-1 SP, D-2 XP, E-2 ES, E-17 EX, E-22 XP, G-5 SP, J-4 SP
<img src="
"/>
<img src="
"/>
colony PCR
H-4
<img src="
"/>
H-4#1,2,3:OK!
ligation - TF
H-5 (G-5 SP + E-22 XP)
J-6 (J-4 SP + E-22 XP)
E-3 (A-1 SP + D-2 XP)
F-1 (E-2 ES + E-17 EX)
9,23,2014
Lab member
Yoshikawa,Nakashima,Nakamura,Tara,Yamanaka
Contents
digestion, gel extraction
A-1CP SP, D-2 XP, E-22 XP, G-5 SP
J-4 SP(stopped)
<img src="
"/>
<img src="
"/>
miniprep
H-4(made glycerol stock)
colony PCR
J-6
<img src="
"/>
OK!
ligation,TF
H-5(G-5 SP+E22 XP)
E-3(A-1 SP+D-2 XP)
9,24,2014
Lab member
Nakashima,Yamanaka,Takemura
Contents
miniprep
F-1,J-6(made glycerol stock)
E-22,D-6
G-6 did not grow.
We disposed J-6.(dropped on the floor)
digestion,gel extraction
A-1 SP,E-2 ES,E-5 ES,E-17 EX,E-18CP EX,E-21 XP,G-5 SP,D-2 XP,F-1 SP,E-19 XP,E-22 XP
<img src="
"/>
<img src="
"/>
ligation,TF
J-2(F-1 SP+E-1 XP)
H-1(F-1 SP+E-21 XP)
F-3(E-5 ES+E-17 EX)
F-4(E-2 ES+E-18 EX)
E-3(A-1 SP+D-2 XP)
H-5(G-5 SP+E-22 XP)
9,25,2014
Lab member
Nakashima,Nakamura,Tara,Tsukada,Yamanaka
Contents
miniprep
A-1CP, J-6
digestion,gel extraction
A-1 SP,D-2 XP,E-5 ES,E-17 EX,E-19 XP,E-21 XP,E-22 XP,F-1 SP,G-5 SP
<img src="
"/>
<img src="
"/>
ligation,TF
J-2(F-1 SP+E-19 XP)
H-1(F-1 SP+E-21 XP)
F-3(E-5 ES+E-17 EX)
E-3(A-1 SP+D-2 XP)
H-5(G-5 SP+E-22 XP)
Making competent cell
preculture
G-5,E-5,E-17,E. coli JM109
competent cell(made at 0911) check
Ampicillin,Chloramphenicol,non-antibiotics
culture in LB 3ml
LB midium 3ml, as N.C.
culture for 2h.
no TF
result of check
Ampicilln,non-antibiotics:become clouded
Chroramphenicol,LB:did not grow
So,we confirmed ampicillin resistant plasmid exists in competent cell and we disposed it.
9,26,2014
Lab member
Yoshikawa,Nakashima,Tara,Yoshikawa(Fresh),Tsukada
Contents
miniprep
G-5,E-5,E-17
digestion
A-1CP SP,D-2 XP,G-5 EP(strange band appeared,disposed),pSB1C3(A-4) EP,A-1 SP
<img src="
"/>
<img src="
"/>
colony PCR
F-3,F-4,H-1,H-5,J-2
<img src="
"/>
<img src="
"/>
OK!
ligation,TF
E-3(D-2 XP+A-1SP)
E-3CP(D-2 XP+A-1CP SP)
preculture
F-3,F-4,H-1,H-5,J-2#1,G-5
9,27,2014
Lab member
Yoshikawa,Nakashima
Contents
TF
E-3(chemical)
miniprep
F-3,F-4,H-1,H-5,J-2(made glycerol stock)
G-5
TF
E-3CP(electroporation)
9,29,2014
Lab member
Yoshikawa,Nakashima,Nakamura,Tara,Takemura
Contents
digestion,gel extraction
A-1CP SP,pSB1C3(A-4) EP,D-2 XP,E-21 XP,G-5 EP,H-5 EP,J-2 SP
<img src="
"/>
<img src="
"/>
Gel making
ligation
G-5CP(G-5 EP+pSB1C3 EP)
H-5CP(H-5 EP+pSB1C3 EP)
J-5(J-2 SP+E-21 XP)
E-3(A-1CP SP+P-2 XP)
preculture
E-5,E-11,E-22
K-1,E-23',Z-1(for assay)
PCR
VF2-(ligation product of E-3)-VR
E-3 lig:1uL
VF2:1uL
VR:1uL
Taq:5uL
MilliQ:2uL
Total:10uL
extention:1m12sec
<img src="
"/>
We observed the band which had expected length.
The ligtion must be OK!
9,30,2014
Lab member
Yoshikawa.Nakamura,Tsukada,Tara,Yamanaka
Contents
miniprep
E-2,E-5,E-11
digestion,gel extraction
We could not find D-2 sample(probably accidentally disposed).
So we stopped it.
colony PCR
G-5,H-5,J-5
<img src="
"/>
H-5,J-5#1,2,3,4:OK!
Making M9 medium
1M MgSO4 50ml
1M CaCl2 10ml
20% glucose 50ml
digestion(cutcheck)
G-5 E,G-5 P,G-5 non-cut
<img src="
"/>
Strange bands appeared.
We decided to read a sequence of this part.
10,1,2014
Lab member
Nakashima,Tara,Nakamura,Takemura
Contens
miniprep
H-5CP,G-5CP,J-5(made glycerol stock)
sequence order
E-2,E-8,E-9,E-19,F-1,F-3,F-4,G-5,H-1,H-4
H-5(VF-VR,Psigma2F-anti2R),J-2,J-4,J-5,J-6
10,2,2014
Lab member
Nakashima,Nakamura,Tara
Contents
assay
completely failed
In M9 medium, Escherichia.coli JM109 did not grow well.(doubling time:1h)
The glycerol stock needed to be put a lot.
culture
F-2,F-3,F-4(for M9 check)
PCR
G-1(F,R),G-4(F,R),G-5(F,R),H-1(F,R),H-4(F,R),H-5(F,R)
Template:1ng/uL,1uL
Making M9 medium
5*M9 24mL
20% Glu 1.2mL
MgSO4 240uL
CaCl2 12uL
Amino acid 10mL
mess up to 1L
PCR check
<img src="
"/>
OK!(except G-5)
PCR
G-1,G-4,H-1,H-4
extension time:6m30sec
EpCAM nested
95C 3min-(-95C 30sec-48C 30sec-72C 3min-)*30-72C 5min-4C
preculture
E-17,E-18,F-1,F-2,F-3,F-4,Z-1(M9)
E-23'(LB)
10,3,2014
Lab member
Nakashima,Tara,Nakamura
Contents
PCR clean up and check
<img src="
"/>
G-1,G-4,H-1,H-4:Band in correct position and unexpected position.
EpCAM:No band
digestion,gel extraction
<img src="
"/>
<img src="
"/>
G-1deg linear EP,G-4deglinear EP,H-1deg linear EP,H-4deg linear EP(A-4)
(deg:degradation tag)
PCR
EpCAM nested
94C 3min-(-94C 30sec-48C 30sec-72C 3min-)*30-72C 5min-4C
<img src="
"/>
No band
gel making
ligation,TF
H-1'(H-1'linear EP+pSB1C3 EP)
H-4'(H-4'linear EP+pSB1C3 EP)
G-1'(G-1'linear EP+pSB1C3 EP)
G-4'(G-4'linear EP+pSB1C3 EP)
PCR
EpCAM nested
95C 3min-(-95C 30sec-46C 30sec-72C 3min-)*30-72C 5min-4C
<img src="
"/>
No band
10,4,2014
Lab member
Yoshikawa
assay1
F-2 in 20mL culture
When OD600=0.516,we put 10% arabinose 20uL.
F-1 in 20mL culture
When OD600=0.514,we put 10% arabinose 20uL.
Z-1 in 20mL culture
When OD600=0.544,we put 10% arabinose 20uL.
O/N culture
Culture in 3mL:disposed(OD600 value did not agrees with each other.)
colony PCR
H-1deg,H-4deg,G-1eg,G-4deg
We confirmed by fluorescence.
preculture
H-1deg,H-4deg#1,G-1eg,G-4deg
10,5,2014
Lab member
Yoshikawa
Contents
miniprep
H-1deg,H-4deg#1,G-1eg,G-4deg
preculture
(M9)
E-15CP,E-17,E-18CP,I-2,F-1,2,3,4,Z-1
10,6,2014
Lab member
Yoshikawa,Nakashima,Tara
Contents
assay1,3
E-15,I-2,F-1,F-2,F-3,F-4,F-17,E-18,Z-1
Measured the OD 600 value.
Except I-2,F-2,the value is more than 1.0.
F-1,F-3,E-17,Z-1(20 fold dilution):Culture in 3mL.
The composition of M9 proved to be wrong,so disposed.
Making M9 medium
5*M9 200mL
20% Glu 10mL
amino acids 10mL
1M MgSO4 2mL
1M CaCl2 100uL
MilliQ 778mL
Total 1000mL
result of OD600
F-3(non-Chloramphenicol)
1h:0.094
2h:0.086
F-3(Chloramphenicol)
1h:0.074
2h:0.073
assay1
(tentative)
We measured the fluorescence of F-1,F-2,Z-1(culture in 20mL).
Ex:501nm
F-2:We observed a peak in 511nm.
F-1,Z-1:No peak
preculture
F-1,F-2,F-3,F-4,E-15CP,I-2,Z-1,E-17,E-18CP(M9 medium)
E-23'(LB medium)
10,7,2014
Lab member
Yoshikawa,Nakashima,Tara,Nakamura
Contents
assay
F-1,F-13,E-17,Z-1,E-15,I-2
culture in M9
except E-15,OD600 is more than 1.0.
E-15:over 0.85
F-2:only 0.3
E-23':over 2.0,culture in LB medium
All samples was cultured in 3 mL.(n=5)
F-1,Z-1:cultured in 20mL(flask)(n=1)
OD600 before subculture
E-15:1.113
E-11:0.933
E-18:1.190
E-23':forgot to measure
F-1:0.927
F-2:0.374
F-3:0.878
F-4:0.992
I-2:0.888
Z-1:1.084
PCR
J-5(F,R),J-6(F,R)
J-5:OK!
extension time
F:1800+200=2000bp,4min
R:1850+200=2050bp,4min
PCR
J-6(F,R)
J-5R
extension time:1min
10,8,2014
Lab member
Yoshikawa,Nakashima,Tara
Contents
making gel
subculture
Escherichia.coliMG1655
100 fold dilution
20mL,flask
PCR clean up and check
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J-6F:low concentration
J-6R:shorter band is OK!
We decided that we made J-5,J-6
as H5deg-positive feedback circuit, and H6deg-positive feedback circuit.
sequence order
G-1deg,G-4deg,H-1deg,H-4deg
TF
(electroporation)
F-1,F-2,F-3,F-4,E-17,E-18CP,Z-1,I-2,E-15
G-1deg,G-4deg,H-1deg,h-4deg,J-4,J-4
also,2mL culture as recovery.
10,9,2014
Lab member
Yoshiakwa,Nakashima,Tara
Contents
digestion,gel extraction
E-19 XP,E-20 XP,H-1deg SP,H-4deg SP
making gel
making plate
Chloramphenicol*10
ligation
J-5deg(E-19XP+H-1degSP)
J-6deg(E-20XP+H-4degSP)
We did not have competent cell of E.coli MG1655, so put it in freezer.
preculture
F-1,E-17,E-15,Z-1,I-2:both in LB medium and M9 medium.
MG1655:LB medium
10,10,2014
Lab member
Yoshikawa,Nakashima,Tara,Yamanaka
assay1,3
I-2,E-15,Z-1,F-1,E-17(made glycerol stock,subculture in 20 fold dilution)
MG1655 did not grow, because we accidentally put antibiotics.
OD600(O/N culture)
E-15:1.795
E-17:1.781
F-1:1.937
I-2:1.886
Z-1:1.780
