Team:UT-Tokyo/Counter/Project/Lab

From 2014.igem.org

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Contents

Favorite Parts

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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
 

Other Pars

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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_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 ?

<img src="Sub_protocol.png" class = "contTitle" />

DNA construction

Transformation

Method
1. Put the competent cell on ice and leave it until dissoloved.
2. Add DNA 10 μL to competent cell 100 μL on 2 ml tube.
3. Place on ice for 30 min.
4. Heatshock at 42℃ for 45 sec.
5. Place the tube on ice for 3 min.
6. Add 1000 μL of LB medium and place tube on 37℃ for 30 min.
7. Plate 1000 μL of the medium and spread well.
8. Incubate the plate on 37℃ over night.

Electrophoresis

Method
1. Add agarose to 50 ml or 25ml of 1xTAE buffer in Erlenmeyer flask per 1 gel plate. The concentration of gel is 1% for over 1kbp of DNA and use 2% to under 1kbp.
2. Wrap the top of Erlenmeyer Flask not to completely seal up.
3. Heat it by microwaves until the solution gets transparent.
4. Pour it into a gel maker and set a comb before gelling.
5. After gelling, remove the comb carefully not to break well.
6. Place the gel into tank for electrophoresis and fill 1xTAE up to over the level of gel.
7. Add 1 μl of 10xLoading buffer into 10 μl of DNA solution and vortex it.
8. Apply 11 μl of sample and 6 μl of ladder solution in each well.
9. Electrophoresis at 100 V for 20-30 min.
10. The band of bromophenol blue reaches 80% of the gel, stop electrophoresis and salvage the gel.
11. Check the band under blue light.

Colony PCR

Method
1. Mix DNA solution and reagent as follows.
(Add10xF-universal Primer and 10xR-Univarsal Primer at a final concentration of 5 μM. Then add 2xGoTaqMix and MillQ.)
2. Dispense it to each PCR tube for 10 μl on ice.
3. Pick up single colony on plate and dip to the tube.
4. Set to Thermal cycler. (95℃ 5 min, 95℃ 30 sec, 53℃ 30 sec, 72℃ 30-180 sec (step 2 - 4 repeated 30 times), 72℃ 3 min)
5.Check the length of DNA by Electrophoresis.

Miniprep

Material
WizardR Plus SV Minipreps DNA Purification System (promega)

Method
1. Pour 3 ml of LB broth to test tube.
2. Pick up a single colony and put it into the tube.
3. Incubate it at 37℃ for 12~16 hours with the tube shaking.
4. Transfer 1.5 ml of culture fluid to 1.5 ml tube.
5. Centrifuge at 15000 rpm for 1 min.
6. Throw away the supernatant
7. add the other 1.0 ml of culture.
8. Centrifuge at 15000 rpm for 1 min.
9. Throw away the supernatant
10. Add 250 μl of Cell-Resuspension Sol and Vortex it until precipitation dissolved.
11. Add 250 μl of Cell-Lysis Sol and invert it not to make bubble.
12. Add 10 μl of Alkaline Protease and invert it not to make bubble
13. Leave it for 3 min at room temperature.
14. Add 350 μl of Neutralization Sol and invert it not to make bubble.
15. Centrifuge at 15000 rpm 10 min.
14. Set a collection tube on column and apply the supernatant to it.
15. Centrifuge at15000 rpm for 1 min.
16. Add 750 μl of Column Wash Sol and Centrifuge for 1 min at 15000 rpm.
17. Remove the flow through.
18. Add 250 μl of Column Wash Sol and Centrifuge for 1 min at 15000 rpm.
19. Remove the flow through.
20. Centrifuge for 2 min at 15000 rpm to dry the column.
21. Transfer the column to another 1.5 ml tube.
22. Add 50 μl of nuclease-free water and leave it for 1 min at room temperature.
23. Centrifuge for 1 min at 15000 rpm.
24. Measure concentration of the flow though liquid.

Digestion

Material
The restriction enzymes (EcoRⅠ, XbaⅠ, SpeⅠ and PstⅠ) are purchased or provided as support for iGEM Japan from Promega.
Method
1. Mix DNA solution and reagent as follows.(unit μl)
2. Incubate for 2 hours at 37℃.
3. Add 2 μl of 10x Loading Buffer and Electrophoresis and Gel Extraction.

Gel extraction

Method
1. After electrophoresis, slice the band and put the gel slice into 1.5 ml tube.
2. Add 750 μl of Membrane Bind Sol and stand at 60℃ (Vortex every 2 min)
3. Apply to column-set collection tube and Centrifuge for 1 min at 15000 rpm.
4. Add 750 μl of Membrane Wash Sol to the column.
5. Centrifuge for 1 min and remove the flow through.
6. Add 500 μl of Membrane Wash Sol to the column.
7. Centrifuge for 1 min and remove the flow through.
8. Centrifuge for 2 min to dry the column.
9. Transfer the column to another 1.5 ml tube.
10. Add 20 μl of nuclease-free water and leave it for 1 min at room temperature.
11. Centrifuge for 1 min at 15000 rpm.
12. Measure concentration of the flow though liquid.

Ligation

Method
1. Calculate the mass ratio of Insert DNA to Vector DNA to equal
2. Mix the DNA solution and reagent as follows.
3. Stand it for 15 min at room temperature.

Assay method

Influence on growth of E. coli

Material
Strain: JM109
Medium: LB

Method
1. Culture O/N.
2. Take 30 uL of overnight culture into 3 mL LB medium (containing 50 ug/mL CP).
3. Incubate the culture at 37℃ and measure OD600 evry a hour.


Sigma factor and its crresponding promoter

Material
Strain: MG1655
Medium: M9

Method
1. Culture O/N.
2. Take 150 uL of overnight culture into 3 mL M9 medium (containing 50 ug/mL CP).
3. Incubate the culture until OD600 reaches the range of 0.45-0.95.
4. Add 3 uL arabinose (10%) to the culture.
5. Incubate the culture 3 hours.
6. Measure GFP fluorescence.

Reset function

Material
Strain: MG1655
Medium: M9

Method
1. Culture O/N.
2. Take 150 uL of overnight culture into 3 mL M9 medium (containing 50 ug/mL CP).
3. Incubate the culture until OD600 reaches the range of 0.5-0.7.
4. Add 3 uL arabinose (10%) to the culture.
5. Incubate the culture 3 hours.
6. Measure GFP fluorescence.
7. Add 100mM IPTG 15 uL.
8. Incubate O/N.
9. Measure GFP fluorescence.


Riboregulator

Material
Strain: JM109
Medium: LB

Method
1. Culture O/N.
2. Take 150 uL of overnight culture into 3 mL M9 medium (containing 50 ug/mL CP).
3. Incubate the culture until OD600 reaches the range of 0.5-0.7.
4. Add 3 uL arabinose (10%) to the culture.
5. Incubate the culture 3 hours.
6. Measure GFP fluorescence.


Positive feedback

Material
Strain: MG1655
Medium: M9

Method
1. Culture O/N.
2. Take 150 uL of overnight culture into 3 mL M9 medium (containing 50 ug/mL CP).
3. Incubate the culture until OD600 reaches the range of 0.5-0.7.
4. Add 3 uL arabinose (10%) to the culture.
5. Culture 10 minute /1 hour.
6. Centrifuge at 3200 rpm for 5 min.
7. Throw away the supernatant.
9. Measure GFP fluorescence.


<img src="Sub_lab_note.png" class = "contTitle" />

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13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31    

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)

  1. 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="IMGP0022_20140722_1.JPG"/>


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

Sprinkling:100uL


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="IMGP0033_20140728_1.JPG"/>
(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="IMGP0034_20140728_2.JPG"/>
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="IMGP0035_20140729_1.JPG"/>
There is band in 1500kbp,so 1500kbp in 0728 may be rest of cutting.

No contamination in Dye.


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.

A-1 SP cut:checked on different times.


colony PCR

B-10(from Plate made in 0729)
1kbp Ladder, 1.5h, 2h, 2.5h, B-10(colony PCR), NC
<img src="IMGP0042_20140730_1.JPG"/>
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="IMGP0043_20140731_1.JPG"/>
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="IMGP0044_20140801_1.JPG"/>
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="IMGP0045_20140804_1.JPG"/>
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="IMGP0046_20140805_1.JPG"/>
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="IMGP0047_20140806_1.JPG"/>
A-6:OK
B-10:Incomplete cut
<img src="IMGP0048_20140806_2.JPG"/>

We chenged restriction enzyme.
B-10 XP
<img src="IMGP0051_20140806_3.JPG"/>
OK!
<img src="IMGP0060_20140806_4.JPG"/>

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="IMGP0061_20140807_1.JPG"/>
<img src="IMGP0062_20140807_2.JPG"/>
All bands are self ligation of backbone.

digestion, gel extraction

A-6 SP, B-10 XP

1kbp Ladder, B-10, A-6
<img src="IMGP0063_20140807_3.JPG"/>

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


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="IMGP0063_20140808_1.JPG"/>
FD buffer is appropriate for SP cut.

digestion, gel extraction

A-6 SP, B-10 XP
<img src="IMGP0064_20140808_2.JPG"/>
<img src="IMGP0065_20140808_3.JPG"/>
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="IMGP0066_20140808_4.JPG"/>
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. 1~4, NC

<img src="IMGP0067_20140811_1.JPG"/>

  1. 5~11,NC

<img src="IMGP0069_20140811_2.JPG"/>

  1. 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="IMGP0068_20140811_3.JPG"/>

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="IMGP0070_20140812_1.JPG"/>
C-6:wrong
A-1:OK!

colony PCR

4-11L
<img src="IMGP0071_20140812_2.JPG"/>
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="IMGP0072_20140813_1.JPG"/>
<img src="IMGP0073_20140813_2.JPG"/>
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="IMGP0074_20140813_3.JPG"/>
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="IMGP0075_20140814_1.JPG"/>
<img src="IMGP0076_20140814_2.JPG"/>
OK!
A-10 XP, 100bp Ladder
<img src="IMGP0077_20140814_3.JPG"/>
OK!(low concentration)

100bp Ladder, pSB1A2(B-10), pSB1A2(B-10), A-7, A-9, B-1
<img src="IMGP0079_20140814_4.JPG"/>
<img src="IMGP0080_20140814_5.JPG"/>
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="IMGP0081_20140814_6.JPG"/>

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="IMGP0082_20140815_1.JPG"/>
<img src="IMGP0083_20140815_2.JPG"/>

colony PCR

A-7 #1~4, A-9 #1~4, A-10 #1~4, B-1 #1~4, D-6 #1~4
<img src="IMGP0085_20140815_3.JPG"/>
A-7 #3,4, A-10 #1,2,4, B-1 #1,2,4:OK!

D-6
<img src="IMGP0087_20140815_4.JPG"/>
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="IMGP0088_20140818_1.JPG"/>

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="IMGP0091_20140818_2.JPG"/>
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="IMGP0100_20140821_1.JPG"/>

colony PCR

A-2, A-3, A-4, 1-21P, NC
<img src="IMGP0101_20140821_2.JPG"/>

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="IMGP0102_20140822_1.JPG"/>

digestion → gel extraction

1-21P SP, D-7 XP, C-6 E, C-6 P, C-6(NC)
<img src="IMGP0103_20140822_2.JPG"/>
<img src="IMGP0104_20140822_3.JPG"/>

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="IMGP0105_20140825_1.JPG"/>
<img src="IMGP0106_20140825_2.JPG"/>

Plate making

Ampicillin*10, Chloramphenicol*20

colony PCR

K-1
<img src="IMGP0107_20140825_3.JPG"/>

  1. 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="IMGP0109_20140826_1.JPG"/>
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="IMGP0108_20140826_2.JPG"/>
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="IMGP0115_20140827_1.JPG"/>
<img src="IMGP0117_20140827_2.JPG"/>
D-3 is a little strange.Contamination?

colony PCR

K-2~5 #1~4

  • 100bp Ladder

<img src="IMGP0118_20140827_3.JPG"/>
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="IMGP0114_20140827_4.JPG"/>
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="IMGP0119_20140828_1.JPG"/>
<img src="IMGP0120_20140828_2.JPG"/>
→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="IMGP0121_20140828_3.JPG"/>
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="IMGP0122_20140829_1.JPG"/>
<img src="IMGP0123_20140829_2.JPG"/>

PCR

B-2, D-9(retry)

check & colony PCR

E-23' #1~4, B-2, D-9
<img src="IMGP0124_20140829_3.JPG"/>
L-1~4
<img src="IMGP0125_20140829_4.JPG"/>
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="IMGP0126_20140901_1.JPG"/>
failed

digestion → gel extraction

L-4 ES, E-6 ES, E-18 EX, L-1~3 ES, K-2~5 EX

<img src="IMGP0127_20140901_2.JPG"/>
<img src="IMGP0129_20140901_3.JPG"/>

Making plate

CP *30

colony PCR

A-7 #1~4, A-9 #1
<img src="IMGP0130_20140901_4.JPG"/>
OK!
D-8 #1~4, E-20 #1~4, F-2 #1~4 (100bp Ladder)
<img src="IMGP0131_20140901_5.JPG"/>
D-8 #1~4, E-20 #1~4,F-2#1,4:OK!
B-1 #1~4
<img src="IMGP0132_20140901_6.JPG"/>
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="IMGP0133_20140902_1.JPG"/>
<img src="IMGP0134_20140902_2.JPG"/>
A-8XP
<img src="IMGP0135_20140902_3.JPG"/>
<img src="IMGP0136_20140902_4.JPG"/>

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="IMGP0137_20140902_5.JPG"/>
<img src="IMGP0138_20140902_6.JPG"/>
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="IMGP0145_20140902_7.JPG"/>
OK!
M-4 #1~4, B-2, D-9, PC(VF2→B-10←VR )
NC, G-5
<img src="IMGP0146_20140902_8.JPG"/>
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="IMGP0144_20140902_9.JPG"/>

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="IMGP0147_20140903_1.JPG"/>
<img src="IMGP0148_20140903_2.JPG"/>

colony PCR

C-4 a, C-5 a, D-1 a, E-17
<img src="IMGP0149_20140903_3.JPG"/>
C-4 a#2,3, C-5 a, D-1 a, E-17:OK!
E-21 b, J-4
<img src="IMGP0150_20140903_4.JPG"/>
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="IMGP0154_20140904_1.JPG"/>
<img src="IMGP0155_20140904_2.JPG"/>

PCR check & clean up

D-9 #1,3,5,7,9
<img src="IMGP0151_20140904_3.JPG"/>
There are bands in #5,7,9
B-2 1,3,5,7,9,11, B-7.
<img src="IMGP0152_20140904_4.JPG"/>
There are bands in B-2 #5,7, B-7.
B-2 4,6, D-9 10,11,12
<img src="IMGP0153_20140904_5.JPG"/>
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="IMGP0156_20140904_6.JPG"/>
D-5, D-6, N-1, N-2 #1,2:OK!
N-3~6 #1~4
<img src="IMGP0157_20140904_7.JPG"/>
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="IMGP0158_20140905_1.JPG"/>
<img src="IMGP0159_20140905_2.JPG"/>

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="IMGP0160_20140905_3.JPG"/>
B-7 #2,3, D-9 #1, E-1 #3,4:OK!
Z-1 #2~4
<img src="IMGP0161_20140905_4.JPG"/>

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


</p>

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="IMGP0162_20140906_1.JPG"/>

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="IMGP0163_20140908_1.JPG"/>
<img src="IMGP0165_20140908_2.JPG"/>
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="IMGP0166_20140908_3.JPG"/>
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="IMGP0167_20140908_4.JPG"/>

colony PCR

E-11,12,14,15 #1~4
<img src="IMGP0168_20140908_5.JPG"/>
OK!
O-3 #1,2
<img src="IMGP0169_20140908_6.JPG"/>

  1. 2:OK!


PCR

B-2
<img src="IMGP0170_20140908_7.JPG"/>
B-7,D-9
<img src="IMGP0171_20140908_8.JPG"/>
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="IMGP0172_20140909_1.JPG"/>
<img src="IMGP0173_20140909_2.JPG"/>

colony PCR

B-2 #1~3, B-2' #1~4, B-7 #1~3, D-9 #1,2, O-1 #1~4
<img src="IMGP0175_20140909_3.JPG"/>
B-2' #1, B-7 #1,2, O-1:OK!
O-2,4,5,6 #1~4
<img src="IMGP0176_20140909_4.JPG"/>
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="IMGP0177_20140910_1.JPG"/>
<img src="IMGP0179_20140910_2.JPG"/>
K-3 EX, K-5 EX, O-1 ES, N-2 ES, N-4 ES
<img src="IMGP0180_20140910_3.JPG"/>
<img src="IMGP0181_20140910_4.JPG"/>
O-5 ES, O-6 ES
<img src="IMGP0182_20140910_5.JPG"/>
<img src="IMGP0183_20140910_6.JPG"/>

colony PCR

I-1 #1~4
<img src="IMGP0185_20140910_7.JPG"/>
B-2(140909) #1~8, D-9 #1~8
<img src="IMGP0184_20140910_8.JPG"/>
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="IMGP0186_20140911_1.JPG"/>
<img src="IMGP0187_20140911_2.JPG"/>
E-15 EP, E-18 EP, E-20 EP, D-9 #1 XP, O-3 ES
<img src="IMGP0188_20140911_3.JPG"/>
<img src="IMGP0189_20140911_4.JPG"/>

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="IMGP0190_20140911_5.JPG"/>
C-1', C-3', C-8, O-2, O-4 #1,2:OK!
P-1, P-5, P-6 #1~3
<img src="IMGP0192_20140911_6.JPG"/>
→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="IMGP0193_20140912_1.JPG"/>
<img src="IMGP0194_20140912_2.JPG"/>
C-1,3:did not extracted
P-1 XP, P-5 XP, P-6 XP
<img src="IMGP0195_20140912_3.JPG"/>
<img src="IMGP0196_20140912_4.JPG"/>

colony PCR

A-1 CP, A-10 CP, C-3, E-6 CP #1~3
<img src="IMGP0198_20140912_5.JPG"/>
E-12 CP, E-15 CP, E-20 CP, E-22a, E-22b
<img src="IMGP0199_20140912_6.JPG"/>
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="IMGP0200_20140915_1.JPG"/>
<img src="IMGP0201_20140915_2.JPG"/>

colony PCR

C-1 (σ20F, VR)
<img src="IMGP0202_20140915_3.JPG"/>
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="IMGP0206_20140916_1.JPG"/>
A-10
<img src="IMGP0207_20140916_2.JPG"/>

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="IMGP0205_20140916_3.JPG"/>

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="IMGP0204_20140916_4.JPG"/>
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="IMGP0208_20140917_1.JPG"/>
<img src="IMGP0209_20140917_2.JPG"/>

colony PCR

D-2, E-18 CP, E-22
<img src="IMGP0218_20140917_3.JPG"/>

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="IMGP0211_20140917_4.JPG"/>
<img src="IMGP0210_20140917_5.JPG"/>
<img src="IMGP0213_20140917_6.JPG"/>
<img src="IMGP0212_20140917_7.JPG"/>
<img src="IMGP0215_20140917_8.JPG"/>
<img src="IMGP0214_20140917_9.JPG"/>
<img src="IMGP0217_20140917_10.JPG"/>
<img src="IMGP0216_20140917_11.JPG"/>

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="IMGP0219_20140918_1.JPG"/>
<img src="IMGP0220_20140918_2.JPG"/>

colony PCR

E-8, E-9 #1~4
<img src="IMGP0221_20140918_3.JPG"/>
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="IMGP0222_20140919_1.JPG"/>
<img src="IMGP0223_20140919_2.JPG"/>

colony PCR

E-2, E-19 #1~4
E-3, H-5 :No colony
<img src="IMGP0225_20140919_3.JPG"/>

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="IMGP0226_20140922_1.JPG"/>
<img src="IMGP0235_20140922_2.JPG"/>

colony PCR

H-4
<img src="IMGP0236_20140922_3.JPG"/>
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="IMGP0241%27.jpg"/>
<img src="IMGP0242%27.jpg"/>

miniprep

H-4(made glycerol stock)

colony PCR

J-6
<img src="IMGP0243%27%27%27.jpg"/>
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="IMGP0244%27.jpg"/>
<img src="IMGP0245%27.jpg"/>

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="IMGP0247%27.jpg"/>
<img src="IMGP0248%27.jpg"/>

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="IMGP0249%27%27.jpg"/>
<img src="IMGP0250%27.jpg"/>

colony PCR

F-3,F-4,H-1,H-5,J-2
<img src="IMGP0251%27.jpg"/>
<img src="IMGP0253%27.jpg"/>
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="IMGP0256%280929%29%27.jpg"/>
<img src="IMGP0257%27.jpg"/>

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="IMGP0258%280929%29%27.jpg"/>
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="IMGP0258%280930%29%27.jpg"/>
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="IMGP0260%27.jpg"/>
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="IMGP0261%27.jpg"/>

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="IMGP0262%27.jpg"/>
G-1,G-4,H-1,H-4:Band in correct position and unexpected position.
EpCAM:No band

digestion,gel extraction

<img src="IMGP0263%27.jpg"/>
<img src="IMGP0264%27.jpg"/>
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="IMGP0265%27.jpg"/>
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="IMGP0267%27.jpg"/>
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

<img src="IMGP0268%27.jpg"/>
<img src="IMGP0269%27.jpg"/>
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

making M9 medium