Team:UT-Tokyo/LabNote
From 2011.igem.org
(Difference between revisions)
Line 377: | Line 377: | ||
*Making Gel | *Making Gel | ||
*Digest(<nowiki>#</nowiki>23 XPcut) | *Digest(<nowiki>#</nowiki>23 XPcut) | ||
- | *IPTG | + | *IPTG induce(<nowiki>#</nowiki>20-9) |
=='11/08/05(Fri)== | =='11/08/05(Fri)== | ||
Line 458: | Line 458: | ||
*Digest(8/17<nowiki>#</nowiki>3,<nowiki>#</nowiki>33 SPcut, <nowiki>#</nowiki>14-5 XPcut, <nowiki>#</nowiki>33 Ecut, <nowiki>#</nowiki>3-27,<nowiki>#</nowiki>3-30,<nowiki>#</nowiki>20-28 EScut, pSB1K3 EPcut) | *Digest(8/17<nowiki>#</nowiki>3,<nowiki>#</nowiki>33 SPcut, <nowiki>#</nowiki>14-5 XPcut, <nowiki>#</nowiki>33 Ecut, <nowiki>#</nowiki>3-27,<nowiki>#</nowiki>3-30,<nowiki>#</nowiki>20-28 EScut, pSB1K3 EPcut) | ||
*Hotstart-PCR (Change conditions a little ) | *Hotstart-PCR (Change conditions a little ) | ||
- | + | **From Genome : uvrBp, cheZ | |
- | + | **From nested-region : uvrA | |
*Culture from 0809 master-plate ⑪ (<nowiki>#</nowiki>2-3-11-5) → Miniprep. | *Culture from 0809 master-plate ⑪ (<nowiki>#</nowiki>2-3-11-5) → Miniprep. | ||
*Making LB plate & tube with various agarose conc. (plate: 0.5, 0.7, 0.9%×2 each, tube: 0.5,1.0,1.5,2.0,3.0%×2 each)(selection: Kanamycin) | *Making LB plate & tube with various agarose conc. (plate: 0.5, 0.7, 0.9%×2 each, tube: 0.5,1.0,1.5,2.0,3.0%×2 each)(selection: Kanamycin) | ||
Line 508: | Line 508: | ||
=='11/08/24(Wed)== | =='11/08/24(Wed)== | ||
- | *Gel extraction & Electrophoresis(<nowiki>#</nowiki>3-27-5_ES, <nowiki>#</nowiki>14-5_XP, <nowiki>#</nowiki>3-27-5_X, *PCR for amplification (or cloning) -> PCR clean-up system -> digest | + | *Gel extraction & Electrophoresis(<nowiki>#</nowiki>3-27-5_ES, <nowiki>#</nowiki>14-5_XP, <nowiki>#</nowiki>3-27-5_X, *PCR for amplification (or cloning) -> PCR clean-up system -> digest |
**<nowiki>#</nowiki>27 (82.1 ng/ul, total = 30 ul) -> XP cut | **<nowiki>#</nowiki>27 (82.1 ng/ul, total = 30 ul) -> XP cut | ||
**<nowiki>#</nowiki>28 (104.4 ng/ul, total = 30 ul) -> XP cut | **<nowiki>#</nowiki>28 (104.4 ng/ul, total = 30 ul) -> XP cut | ||
Line 534: | Line 534: | ||
*Colony PCR (<nowiki>#</nowiki>33 Miniprep transformations) | *Colony PCR (<nowiki>#</nowiki>33 Miniprep transformations) | ||
*Gel extraction (<nowiki>#</nowiki>14 XPcut) | *Gel extraction (<nowiki>#</nowiki>14 XPcut) | ||
- | *Ligation (retry) (using today's competent cells & 0802 | + | *Ligation (retry) (using today's competent cells & 0802 ΔcheZ cells) |
*Cloning (cheZ) | *Cloning (cheZ) | ||
*Digest (<nowiki>#</nowiki>5 EXcut, cheZ PCR products XPcut) | *Digest (<nowiki>#</nowiki>5 EXcut, cheZ PCR products XPcut) | ||
Line 547: | Line 547: | ||
=='11/08/28(Sun)== | =='11/08/28(Sun)== | ||
- | *Making | + | *Making ΔcheZ comp., LB amp plates, TB |
*Colony PCR & master plate preparation -> Failure!! | *Colony PCR & master plate preparation -> Failure!! | ||
*Gel extraction (<nowiki>#</nowiki>29 XPcut, <nowiki>#</nowiki>5 EXcut) | *Gel extraction (<nowiki>#</nowiki>29 XPcut, <nowiki>#</nowiki>5 EXcut) | ||
Line 556: | Line 556: | ||
=='11/08/29(Mon)== | =='11/08/29(Mon)== | ||
*PCR purification -> digest with XP | *PCR purification -> digest with XP | ||
- | **<nowiki>#</nowiki>27(251.3ng/ul),<nowiki>#</nowiki>28 | + | **<nowiki>#</nowiki>27(251.3ng/ul),<nowiki>#</nowiki>28 |
*Miniprep | *Miniprep | ||
- | **<nowiki>#</nowiki>20(128.1ng/ul), <nowiki>#</nowiki>14-5 | + | **<nowiki>#</nowiki>20(128.1ng/ul), <nowiki>#</nowiki>14-5 |
*Gel extraction(<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>20) | *Gel extraction(<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>20) | ||
*Ligation (higher I/V ratio, into cheZ comp.)(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5(retry), <nowiki>#</nowiki>2-3-11-5 to pSB1K3 (retry), <nowiki>#</nowiki>35-pSB1AK3, (<nowiki>#</nowiki>33,<nowiki>#</nowiki>34)-<nowiki>#</nowiki>3-17-5) | *Ligation (higher I/V ratio, into cheZ comp.)(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5(retry), <nowiki>#</nowiki>2-3-11-5 to pSB1K3 (retry), <nowiki>#</nowiki>35-pSB1AK3, (<nowiki>#</nowiki>33,<nowiki>#</nowiki>34)-<nowiki>#</nowiki>3-17-5) | ||
Line 597: | Line 597: | ||
**Titer check(<nowiki>#</nowiki>3-17-5(Takara comp.,1:9gradient), <nowiki>#</nowiki>2-3-11-5(ΔcheZ comp.0802, 1:9gradient), <nowiki>#</nowiki>2-3-11-5(ΔcheZ comp.0830, 1:9gradient)) | **Titer check(<nowiki>#</nowiki>3-17-5(Takara comp.,1:9gradient), <nowiki>#</nowiki>2-3-11-5(ΔcheZ comp.0802, 1:9gradient), <nowiki>#</nowiki>2-3-11-5(ΔcheZ comp.0830, 1:9gradient)) | ||
**Ligation products | **Ligation products | ||
+ | |||
+ | =='11/09/01(Thu)== | ||
+ | *Colony PCR | ||
+ | **<nowiki>#</nowiki>34-3-17-5(MP), <nowiki>#</nowiki>3-14, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>35-pSB1AK3, <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>37(from Part cloning) | ||
+ | *Gel ext. | ||
+ | **<nowiki>#</nowiki>27XP ,<nowiki>#</nowiki>28XP(0830); <nowiki>#</nowiki>27XP, <nowiki>#</nowiki>28XP, <nowiki>#</nowiki>3-30XP -> failed!, <nowiki>#</nowiki>14-5XP(0831) | ||
+ | *Making medium(SOB, LB amp plate) | ||
+ | *Making competent cells (WT & ΔcheZ) | ||
+ | *Miniprep(<nowiki>#</nowiki>3, <nowiki>#</nowiki>20) | ||
+ | *PCR & Column purification | ||
+ | **<nowiki>#</nowiki>3-14, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>3-14-5 (Recovery from colony PCR tubes!) | ||
+ | *Digest | ||
+ | **<nowiki>#</nowiki>3 -> Failed (Mixed with DMSO!), <nowiki>#</nowiki>20 | ||
+ | **<nowiki>#</nowiki>3-14, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>3-14-5 | ||
+ | *Ligation | ||
+ | **<nowiki>#</nowiki>3-27, <nowiki>#</nowiki>20-<nowiki>#</nowiki>3-17-5 | ||
+ | *Transformation | ||
+ | **<nowiki>#</nowiki>36 into Nippon Gene comp. | ||
+ | **Ligation products into Takara comp. (including N.C.) | ||
+ | **Titer check(MP product <nowiki>#</nowiki>3 into 0901 WT comp., MP product <nowiki>#</nowiki>20 into 0901 ΔcheZ comp.) | ||
+ | *Mobility assay | ||
+ | **<nowiki>#</nowiki>14-5 (km resistance) WT/ΔcheZ cells | ||
+ | |||
+ | =='11/09/02(Fri)== | ||
+ | *Making medium and reagent(LB 120ml(0.125, 0.25, 0.5% Agar, M9 stock(20×), M9 200ml, Vitamins Solution (100×)) | ||
+ | *Gel extraction -> Digest products were OK but extraction was failed. | ||
+ | **<nowiki>#</nowiki>20_SP, <nowiki>#</nowiki>3-14_XP, <nowiki>#</nowiki>3-14-5_XP, <nowiki>#</nowiki>3-29_XP | ||
+ | *Colony PCR & Master plate(<nowiki>#</nowiki>20-3-17-5, <nowiki>#</nowiki>3-27) | ||
+ | *Miniprep(<nowiki>#</nowiki>3, <nowiki>#</nowiki>20, <nowiki>#</nowiki>37, <nowiki>#</nowiki>3-14, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>20-3-17-5, <nowiki>#</nowiki>3-27, ΔcheZ) | ||
+ | *Hot-start PCR & Column purification -> PCR products were OK but all purified products were lost. | ||
+ | **<nowiki>#</nowiki>20-3-17-5, <nowiki>#</nowiki>3-27 (Recovery from colony PCR tubes) | ||
+ | **<nowiki>#</nowiki>3-14, <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-29 (from miniprep products) | ||
+ | *PCR & Column purification | ||
+ | **<nowiki>#</nowiki>20-3-17-5, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-29 (from miniprep products) | ||
+ | *Digest | ||
+ | **<nowiki>#</nowiki>3_SP, <nowiki>#</nowiki>20_SP, <nowiki>#</nowiki>37_ES, <nowiki>#</nowiki>3-14_ES, <nowiki>#</nowiki>3-29_ES, <nowiki>#</nowiki>3-14-5_XP, ΔcheZ?_E/P (from MP products) | ||
+ | **<nowiki>#</nowiki>20-3-17-5_XP, <nowiki>#</nowiki>3-27_ES, <nowiki>#</nowiki>3-14-5_XP, <nowiki>#</nowiki>3-29_ES (from PCR products) | ||
+ | |||
+ | =='11/09/03(Sat)== | ||
+ | *Gel extraction | ||
+ | *Ligation & Transformation | ||
+ | **<nowiki>#</nowiki>20-<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>2-<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-27-<nowiki>#</nowiki>5 into Nippon Gene comp. (+ High Competent Broth 240ul) | ||
+ | **<nowiki>#</nowiki>3-27-<nowiki>#</nowiki>5, <nowiki>#</nowiki>37-<nowiki>#</nowiki>5 into 0902 WT comp.(+ Takara SOC broth 500ul) | ||
+ | *Assays | ||
+ | **Mobility assay (.5%, .25%, .125%) | ||
+ | **Asp assay | ||
+ | *Part Cloning (<nowiki>#</nowiki>36) | ||
+ | |||
+ | =='11/09/04(Sun)== | ||
+ | *Hot-start PCR Pre-Mix (4×) preparation (Buffer + dNTPs + THUNDER Taq) | ||
+ | *Colony PCR(<nowiki>#</nowiki>20-3-14-5, <nowiki>#</nowiki>3-27-5, <nowiki>#</nowiki>3-29-5, <nowiki>#</nowiki>37-5) | ||
+ | *Colony Diffusion Assays | ||
+ | *GFP Live-Imaging Assays | ||
+ | *Ligation & Transformation (retry) | ||
+ | **<nowiki>#</nowiki>2-<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-27-<nowiki>#</nowiki>5, <nowiki>#</nowiki>3-29-<nowiki>#</nowiki>5, <nowiki>#</nowiki>37-<nowiki>#</nowiki>5, <nowiki>#</nowiki>20-<nowiki>#</nowiki>28 into 0901 WT comp. | ||
+ | **<nowiki>#</nowiki>36 into Takara comp. | ||
+ | **<nowiki>#</nowiki>2, <nowiki>#</nowiki>5 as Negative Controls into 0901 WT comp. | ||
+ | *Culture for Miniprep(<nowiki>#</nowiki>20-3-14-5, <nowiki>#</nowiki>20) | ||
+ | *Digest(<nowiki>#</nowiki>20) | ||
+ | |||
+ | =='11/09/05(Mon)== | ||
+ | *Colony Diffusion Assays (<nowiki>#</nowiki>2-9) WT,cheZ- | ||
+ | *Gel extraction(<nowiki>#</nowiki>20) | ||
+ | *Luciferase Assays | ||
+ | **Calibrations (Firefly Luciferase standards) | ||
+ | **Background Assay | ||
+ | **Dual Luciferase Assay(<nowiki>#</nowiki>20-3-14-5, <nowiki>#</nowiki>20-3-17-5) | ||
+ | *Colony PCR(<nowiki>#</nowiki>2-<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-27-<nowiki>#</nowiki>5, <nowiki>#</nowiki>3-29-<nowiki>#</nowiki>5, <nowiki>#</nowiki>37-<nowiki>#</nowiki>5, <nowiki>#</nowiki>20-<nowiki>#</nowiki>28, <nowiki>#</nowiki>36) | ||
+ | *PCR -> clean-up -> Digest(<nowiki>#</nowiki>3-29-<nowiki>#</nowiki>5, <nowiki>#</nowiki>37-5) | ||
+ | *IPTG-inducted Cell lysis test(IPTG+, LB plate (Positive control)) | ||
+ | *Miniprep(<nowiki>#</nowiki>20-3-14-5, <nowiki>#</nowiki>37-5, <nowiki>#</nowiki>36) | ||
+ | *Making M9 -> Asp -> all failed(.125%, .25% Agar) | ||
+ | *Culture for Miniprep(<nowiki>#</nowiki>2-3-14-5) | ||
+ | |||
+ | =='11/09/06(Tue)== | ||
+ | *Gel extraction(<nowiki>#</nowiki>37-5, <nowiki>#</nowiki>3-29-5) | ||
+ | *Miniprep(<nowiki>#</nowiki>2-3-14-5, <nowiki>#</nowiki>20-3-14-5, <nowiki>#</nowiki>20-3-17-5, <nowiki>#</nowiki>36, <nowiki>#</nowiki>3-29-5, <nowiki>#</nowiki>37-5) | ||
+ | *PCR (retry) (Hot-start & Normal PCR) -> Purification -> Digest(<nowiki>#</nowiki>3-29-5_XP, <nowiki>#</nowiki>37-5_ES) | ||
+ | *PCR (cloning retry)(lexA, uvrBp) | ||
+ | *Ligation & Transformation | ||
+ | **<nowiki>#</nowiki>3-27-<nowiki>#</nowiki>5, <nowiki>#</nowiki>3-30-<nowiki>#</nowiki>5, <nowiki>#</nowiki>2-3-11-5-pSB1AK3 | ||
+ | **<nowiki>#</nowiki>5, pSB1AK3 (N.C.) | ||
+ | *Culture for GFP live-imaging (<nowiki>#</nowiki>2-9 WT/cheZ-) -> 4℃ storage | ||
+ | *Ninhydrin sol. preparation (Not dissolvable!) -> 4℃ storage | ||
+ | |||
+ | =='11/09/07(Wed)== | ||
+ | *Colony PCR(<nowiki>#</nowiki>3-27-5, <nowiki>#</nowiki>3-30-5) | ||
+ | *Digest(<nowiki>#</nowiki>36_SP) | ||
+ | *Culture from master plate(<nowiki>#</nowiki>36, <nowiki>#</nowiki>3-29-5, <nowiki>#</nowiki>37-5) | ||
+ | *Miniprep(<nowiki>#</nowiki>36, <nowiki>#</nowiki>3-29-5, <nowiki>#</nowiki>37-5, <nowiki>#</nowiki>3-27-5 (failed)) | ||
+ | *PCR purification & Digest(lexA_XP, uvrBp_ES) | ||
+ | *Gel extraction(<nowiki>#</nowiki>3-29-5_XP, <nowiki>#</nowiki>37-5_ES, pSB1AK3_EP, <nowiki>#</nowiki>36_SP) | ||
+ | *PCR amplification -> Digest(<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-29-5, <nowiki>#</nowiki>37-5, <nowiki>#</nowiki>3-27-5) | ||
+ | *Digest(<nowiki>#</nowiki>36_SP (re), <nowiki>#</nowiki>2_SP, <nowiki>#</nowiki>2-3-17-5_EX, <nowiki>#</nowiki>3-14-5_ES, <nowiki>#</nowiki>34_SP) | ||
+ | *Culture for Miniprep(<nowiki>#</nowiki>2, <nowiki>#</nowiki>34, <nowiki>#</nowiki>36, <nowiki>#</nowiki>3-27-5, <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>2-3-17-5) | ||
+ | Ligation & Transformation | ||
+ | **<nowiki>#</nowiki>33-<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>34-<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>36-<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>2-<nowiki>#</nowiki>3-29-5, <nowiki>#</nowiki>20-<nowiki>#</nowiki>3-29-5, <nowiki>#</nowiki>3-<nowiki>#</nowiki>37-5, <nowiki>#</nowiki>2-3-11-5-pSB1AK3, <nowiki>#</nowiki>1-<nowiki>#</nowiki>3-17-5 | ||
+ | **<nowiki>#</nowiki>33, <nowiki>#</nowiki>34, <nowiki>#</nowiki>36, <nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>20, <nowiki>#</nowiki>3, pSB1AK3 (N.C.) | ||
+ | *Ninhydrin stock solution preparation (10%, H2O : EtOH = 9:1) | ||
+ | **Asp conc. check (it took 5 min at 80C) | ||
+ | *Making M9 medium(.25, .125% Agar.) | ||
+ | |||
+ | =='11/09/08(Thu)== | ||
+ | *Making competent cell (WT), LB(amp) plate | ||
+ | *Frozen stocks(<nowiki>#</nowiki>36, <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-27-5) | ||
+ | *Colony PCR(<nowiki>#</nowiki>33-3-14-5, <nowiki>#</nowiki>34-3-14-5, <nowiki>#</nowiki>36-3-14-5, <nowiki>#</nowiki>20-3-29-5, <nowiki>#</nowiki>2-3-11-5(psB1AK3)) | ||
+ | *Miniprep(<nowiki>#</nowiki>2, <nowiki>#</nowiki>34, <nowiki>#</nowiki>36, <nowiki>#</nowiki>2-3-17-5, <nowiki>#</nowiki>3-27-5, <nowiki>#</nowiki>3-14-5) | ||
+ | *Gel extraction | ||
+ | **PCR product: <nowiki>#</nowiki>35_SP, <nowiki>#</nowiki>31_XP, <nowiki>#</nowiki>3-14-5_XP, <nowiki>#</nowiki>3-27-5_XP, <nowiki>#</nowiki>3-29-5_XP, <nowiki>#</nowiki>37-5_XP | ||
+ | **Miniprep product: <nowiki>#</nowiki>2_SP, <nowiki>#</nowiki>34_SP, <nowiki>#</nowiki>36_SP, <nowiki>#</nowiki>2-3-17-5_EX, <nowiki>#</nowiki>3-14-5_ES | ||
+ | *PCR amplification(<nowiki>#</nowiki>34-3-14-5, <nowiki>#</nowiki>36-3-14-5, <nowiki>#</nowiki>20-3-29-5, <nowiki>#</nowiki>20-3-14-5) | ||
+ | *PCR for Sequencing -> Store at -20C(<nowiki>#</nowiki>33-3-11-5, <nowiki>#</nowiki>34-3-14-5, <nowiki>#</nowiki>20-3-29-5, <nowiki>#</nowiki>37-5) | ||
+ | *Ligation(<nowiki>#</nowiki>35_ES-psB1AK3_ES, <nowiki>#</nowiki>31_XP-psB1A2_XP, <nowiki>#</nowiki>31_XP-psB1AK3_XP, <nowiki>#</nowiki>3-<nowiki>#</nowiki>37-5, <nowiki>#</nowiki>36-3-29-5, <nowiki>#</nowiki>3-14-5_ES-<nowiki>#</nowiki>2-3-17-5_EX, <nowiki>#</nowiki>20-<nowiki>#</nowiki>3-27-5) | ||
+ | *Culture for Miniprep(<nowiki>#</nowiki>34-3-17-5, <nowiki>#</nowiki>36-3-14-5, <nowiki>#</nowiki>20-3-29-5, <nowiki>#</nowiki>2-3-11-5) | ||
+ | *Digest | ||
+ | **PCR products: <nowiki>#</nowiki>34-3-14-5_ES, <nowiki>#</nowiki>36-3-14-5_ES, <nowiki>#</nowiki>20-3-14-5_ES | ||
+ | **pSB1C3_EP | ||
+ | *Asp Gradient test | ||
+ | |||
+ | =='11/09/09(Fri)== | ||
+ | *Making competent cell (WT), TB | ||
+ | *Colony PCR(<nowiki>#</nowiki>35-pSB1AK3 (failed), <nowiki>#</nowiki>31-pSB1AK3, <nowiki>#</nowiki>3-<nowiki>#</nowiki>37-5, <nowiki>#</nowiki>36-<nowiki>#</nowiki>3-29-5, <nowiki>#</nowiki>3-14-5-<nowiki>#</nowiki>2-3-17-5, <nowiki>#</nowiki>20-3-27-5(failed)) | ||
+ | *PCR amplification | ||
+ | **From Miniprep : <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-17-5 | ||
+ | **From Colony PCR : <nowiki>#</nowiki>31, <nowiki>#</nowiki>3-37-5, <nowiki>#</nowiki>36-3-29-5, <nowiki>#</nowiki>3-14-5-2-3-17-5 (failed) | ||
+ | *Digest | ||
+ | **Miniprep product: <nowiki>#</nowiki>3-11-5_EP | ||
+ | **PCR-purified product: <nowiki>#</nowiki>3-14-5_EP, <nowiki>#</nowiki>3-17-5_EP, <nowiki>#</nowiki>31_EP, <nowiki>#</nowiki>3-37-5_XP, <nowiki>#</nowiki>36-3-29-5_ES | ||
+ | *Miniprep(<nowiki>#</nowiki>34-3-14-5, <nowiki>#</nowiki>36-3-14-5, <nowiki>#</nowiki>20-3-29-5, <nowiki>#</nowiki>2-3-11-5 (pSB1AK3)) | ||
+ | *Gel extraction(<nowiki>#</nowiki>34-3-14-5_ES, <nowiki>#</nowiki>36-3-14-5_ES, <nowiki>#</nowiki>20-3-14-5_ES, <nowiki>#</nowiki>3-11-5_EP) | ||
+ | *Ligation | ||
+ | **<nowiki>#</nowiki>3-11-5_EP-pSB1C3_EP, <nowiki>#</nowiki>29_XP-pSB1AK3_XP | ||
+ | **<nowiki>#</nowiki>36-3-14-5_ES-<nowiki>#</nowiki>2-3-17-5_EX, <nowiki>#</nowiki>20_SP-<nowiki>#</nowiki>3-27-5_XP(retry) | ||
+ | *Comp. check | ||
+ | **<nowiki>#</nowiki>2-3-11-5 (pSB1AK3) into 0908 WT comp. | ||
+ | **<nowiki>#</nowiki>20-3-29-5 into 0909 WT comp. | ||
+ | **<nowiki>#</nowiki>20-3-29-5 into 0802 cheZ- comp. | ||
+ | *Part cloning(BBa_J04450 (IPTG-induced RFP reporter on pSB1C3)) | ||
+ | *Culture for Miniprep(<nowiki>#</nowiki>31-pSB1AK3, <nowiki>#</nowiki>3-37-5, <nowiki>#</nowiki>36-3-29-5, <nowiki>#</nowiki>3-14-5-2-3-17-5) | ||
+ | *UV irradiation -> Luciferase Assay(<nowiki>#</nowiki>24-3-17-5, <nowiki>#</nowiki>34-3-17-5) | ||
+ | *Asp Gradient test | ||
+ | *CleanSEQ -> Sequencing(<nowiki>#</nowiki>33-3-11-5, <nowiki>#</nowiki>34-3-14-5, <nowiki>#</nowiki>20-3-29-5, <nowiki>#</nowiki>37-5) | ||
+ | |||
+ | =='11/09/10(Sat)== | ||
+ | *Culture plate storage (4℃) | ||
+ | *Miniprep(<nowiki>#</nowiki>31-pSB1AK3, <nowiki>#</nowiki>3-37-5, <nowiki>#</nowiki>36-3-29-5, <nowiki>#</nowiki>3-14-5-2-3-17-5) | ||
+ | *Gel extraction(<nowiki>#</nowiki>3-14-5_EP, <nowiki>#</nowiki>3-17-5_EP, <nowiki>#</nowiki>31_EP, <nowiki>#</nowiki>3-37-5_XP, <nowiki>#</nowiki>36-3-29-5_ES) | ||
+ | *Colony PCR(<nowiki>#</nowiki>36-3-14-5-<nowiki>#</nowiki>2-3-17-5 (Maybe Ligation was FAILURE , estimated length is 1000-1500 by electrophoresis)) | ||
+ | *Ligation(<nowiki>#</nowiki>20_SP-<nowiki>#</nowiki>3-37-5_XP) | ||
+ | |||
+ | =='11/09/11(Sun)== | ||
+ | *Digest from Miniprep products | ||
+ | **<nowiki>#</nowiki>3-14-5-2-3-17-5_XP | ||
+ | **<nowiki>#</nowiki>36-3-14-5_SP, <nowiki>#</nowiki>20_SP -> O.N. | ||
+ | *Colony PCR | ||
+ | **<nowiki>#</nowiki>3-11-5 (pSB1C3), <nowiki>#</nowiki>29 (pSB1AK3), BBa_J04450 (pSB1C3) | ||
+ | **<nowiki>#</nowiki>20-<nowiki>#</nowiki>3-27-5(failed), <nowiki>#</nowiki>20-<nowiki>#</nowiki>3-37-5, <nowiki>#</nowiki>36-<nowiki>#</nowiki>3-14-5-2-3-17-5 | ||
+ | *Gel extraction(<nowiki>#</nowiki>3-14-5-2-3-17-5_XP) | ||
+ | *Culture for Miniprep | ||
+ | **From Master plate : <nowiki>#</nowiki>20-3-37-5, <nowiki>#</nowiki>29 (pSB1AK3), BBa_J04450 (pSB1C3) | ||
+ | **From plate stock : <nowiki>#</nowiki>20 | ||
+ | **From frozen stock : <nowiki>#</nowiki>2-3-17-5 | ||
+ | *Miniprep( <nowiki>#</nowiki>36-3-14-5-2-3-17-5) | ||
+ | *Ligation(<nowiki>#</nowiki>20_SP-<nowiki>#</nowiki>3-14-5-2-3-17-5_XP, <nowiki>#</nowiki>1_SP-<nowiki>#</nowiki>3-14-5-2-3-17-5_XP) | ||
+ | *Direct Ligation | ||
+ | **i.PCR amplification -> Digest for >1h -> Heat kill for 20min (<nowiki>#</nowiki>20-3-37-5_XP, <nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES) | ||
+ | **ii.Digest for >1h -> Heat kill for 20min (<nowiki>#</nowiki>36-3-14-5-2-3-17-5_SP, pSB1A3_EP, <nowiki>#</nowiki>2-3-17-5_EX) | ||
+ | **iii.Ligation for 30min (<nowiki>#</nowiki>20-3-14-5_ES-<nowiki>#</nowiki>2-3-17-5_EX, <nowiki>#</nowiki>36-3-14-5-2-3-17-5_SP-<nowiki>#</nowiki>20-3-37-5_XP, <nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES-<nowiki>#</nowiki>20-3-37-5_XP-pSB1A3_EP) | ||
+ | **iv.Transformation | ||
+ | *Making 0.125% agar LB amp plate (IPTG 1, 10, 30, 100uM) | ||
+ | *Cell Diffusion Assay | ||
+ | **<nowiki>#</nowiki>20-3-29-5 WT (IPTG 1, 10, 30, 100uM) | ||
+ | **<nowiki>#</nowiki>20-3-29-5 cheZ-/- (IPTG 1, 10, 30, 100uM) | ||
+ | **WT | ||
+ | **cheZ-/- | ||
=='11/9/12 (Mon)== | =='11/9/12 (Mon)== | ||
Line 659: | Line 834: | ||
**<nowiki>#</nowiki>1-3-14-5-2-3-17-5 | **<nowiki>#</nowiki>1-3-14-5-2-3-17-5 | ||
**<nowiki>#</nowiki>20-3-37-5 (from a non-red colony on the master plate) | **<nowiki>#</nowiki>20-3-37-5 (from a non-red colony on the master plate) | ||
- | *Gel | + | *Gel extraction |
**<nowiki>#</nowiki>20-3-37-5_XP (from non-red colony) | **<nowiki>#</nowiki>20-3-37-5_XP (from non-red colony) | ||
**<nowiki>#</nowiki>20-3-29-5_ES(failed) | **<nowiki>#</nowiki>20-3-29-5_ES(failed) | ||
Line 678: | Line 853: | ||
*Ligation | *Ligation | ||
**<nowiki>#</nowiki>20_SP-<nowiki>#</nowiki>3-27-5_XP | **<nowiki>#</nowiki>20_SP-<nowiki>#</nowiki>3-27-5_XP | ||
- | *Transformation | + | *Transformation |
**<nowiki>#</nowiki>3-14-5-2-3-17-5 | **<nowiki>#</nowiki>3-14-5-2-3-17-5 | ||
**<nowiki>#</nowiki>1-3-14-5-2-3-17-5 | **<nowiki>#</nowiki>1-3-14-5-2-3-17-5 | ||
**<nowiki>#</nowiki>20-3-14-5-2-3-17-5 (for plate stock) | **<nowiki>#</nowiki>20-3-14-5-2-3-17-5 (for plate stock) | ||
**<nowiki>#</nowiki>20-3-37-5 (from a non-red colony on the master plate) (for plasmid check) | **<nowiki>#</nowiki>20-3-37-5 (from a non-red colony on the master plate) (for plasmid check) | ||
+ | |||
+ | =='11/09/14(Wed)== | ||
+ | *Colony PCR | ||
+ | **<nowiki>#</nowiki>3-27-5_XP-<nowiki>#</nowiki>20_SP (failed) | ||
+ | *Culture for Luciferase Assay (from plate stocks) | ||
+ | **<nowiki>#</nowiki>20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM) | ||
+ | **<nowiki>#</nowiki>1-3-14-5-2-3-17-5 (IPTG 0uM) | ||
+ | *Digest | ||
+ | **<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-17-5, <nowiki>#</nowiki>3-14-5-2-3-17-5, <nowiki>#</nowiki>1-3-14-5-2-3-17-5, <nowiki>#</nowiki>20-3-14-5-2-3-17-5 (EPcut) | ||
+ | **<nowiki>#</nowiki>20_SP | ||
+ | *Culture for Miniprep. | ||
+ | **From plate stock : <nowiki>#</nowiki>20, <nowiki>#</nowiki>20-3-14-5-2-3-17-5, <nowiki>#</nowiki>3-14-5-2-3-17-5, <nowiki>#</nowiki>3-17-5 | ||
+ | **From 09/11 master plate : <nowiki>#</nowiki>3-11-5 (pSB1C3) | ||
+ | *Part cloning(<nowiki>#</nowiki>19: BBa_R0010) | ||
+ | *Ligation | ||
+ | **<nowiki>#</nowiki>20_SP-<nowiki>#</nowiki>3-29-5_XP | ||
+ | **<nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES-<nowiki>#</nowiki>20-3-37-5_XP-pSB1C3 | ||
+ | *Making WT competent cell | ||
+ | *Dual Luciferase Assay | ||
+ | **<nowiki>#</nowiki>1-3-14-5-2-3-17-5 (IPTG 0uM) | ||
+ | **<nowiki>#</nowiki>20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM) | ||
+ | *Asp TLC Assay | ||
+ | *Asp Diffusion Assay (0.25% Agar) | ||
+ | *Fumaric Acid (Fum) Sol. preparation | ||
+ | *Sequencing | ||
+ | **①F⑤R<nowiki>#</nowiki>20-3-37-5 (from a non-red colony on the master plate) | ||
+ | **②F⑥R<nowiki>#</nowiki>20-3-29-5 (<nowiki>#</nowiki>2-3-11-5?) | ||
+ | **③F⑦R<nowiki>#</nowiki>3-27-5 | ||
+ | **④F⑧R<nowiki>#</nowiki>31 | ||
<html> | <html> |
Revision as of 02:25, 15 September 2011
Lab Note
![logo](https://static.igem.org/mediawiki/2011/5/5c/UT-Tokyo_logo.png)
iGEM UT-Tokyo
Parts List
Number | Part | Content | Plasmid | Length (bp) |
---|---|---|---|---|
#1 | J23119 | c.Promoter (Strong) | pSB1A2 | 35 |
#2 | J23118 | c.Promoter (Medium) | BBa_J61002 | 35 |
#3 | B0032 | RBS | pSB1A2 | 13 |
#5 | B0014 | d.Ter | pSB1AK3 | 95 |
#9 | E0240 | RBS-GFP-d.Ter | pSB1A2 | 876 |
#10 | E0040 | GFP | pSB1A2 | 681 |
#11 | E1010 | RFP | pSB2K3 | 723 |
#14 | I712019 | fLuc | pSB1AK8 | 1653 |
#17 | J52008 | rLuc | pSB1AK3 | 936 |
#20 | R0011 | lacP | pSB1A2 | 55 |
#21 | C0012 | LacI | pSB1A2 | 1128 |
#22 | I712074 | pT7 | pSB1AK8 | 46 |
#23 | K145001 | T7 RNA Pol. | pSB1A2 | 2655 |
#24 | J22106 | recAp | pSB1A2 | 192 |
#27 | C0083 | AspA | pSB2K3 | 1518 |
#28 | K112808 | T4 phage lysis device (no promoter) | pSB1A2 | 1785 |
#29 | - | CheZ | pSB1AK3 | 728 |
#30 | K117000 | Lysis gene | pSB1A2 | 144 |
#31 | - | LexA | pSB1AK3 | 750 |
#33 | - | sulAp | pSB1AK3 | 67 |
#34 | - | uvrAp | pSB1AK3 | 96 |
#35 | - | recNp | ||
#36 | R0051 | cI-repressed promoter | pSB1A2 | 49 |
#37 | C0051 | cI repressor (LVA tagged) | pSB1A2 | \ |
#38 | - | RecA |
Lab Diary
For convenience sake, each part (genes, promotors, etc) is represented by consecutive numbers (e.g. #20 for lac promotor). By pointing the mouse on the part number in the construct, you can find out the details of the part.
- May
- June
- July
- August
- September
- October
Please enable Javascript to view this calendar.
'11/5/18 (Wed)
- Making LB medium, 50×TAE, Tris-HCl (pH8.0)
'11/5/24 (Tue)
- Making SOB medium, 0.5M EDTA (pH8.0)
'11/5/25(Wed)
- Making TB(pH=6.7), LB plate
'11/5/26(Thu)
- Making Mgaq(for SOB medium), Competent cell
- TB filtration
'11/5/31(Tue)
- iGEM parts resuspension + frozen stock (at -20℃)
- Transformation
- Overnight culture on LB plate (with 100ug/ml ampicilline)
'11/06/01(Wed)
- Picking up colony and transfer to LB medium
- Making LB medium
'11/06/02(Thu)
- Miniprep
- Making Glycerol(50%)(for cryopreservation)
'11/06/07(Tue)
- Nanodrop
- Transformation
- Culture from frozen stock
'11/06/08(Wed)
- Picking up colony
'11/06/09(Thu)
- Miniprep
'11/06/13(Mon)
- Planting Negative control
- Making frozen stock
- Transformation(BBa_E0240,B0032,B0015,B0014,E0040,J31000,J44000)
'11/06/14(Tue)
- Picking up colony
- Making Mg reagent
'11/06/15(Wed)
- Miniprep
'11/06/17(Fri)
- Picking up colony(B0032,B0015,B0040,E0240,J31000,J44000)
- Miniprep(B0014)
- Dissolution(J23119,J23118,K16500,J22106)
'11/06/21(Tue)
- Making frozen stock(B0032,B0015,B0040,E0240,J31000,J44000)
- Passafe culture(E0240,J31000)
- Nanodrop again
'11/06/22(Wed)
- Miniprep(E0240,J31000)
- Making Competent cell
'11/06/24(Fri)
- Digest (product of Miniprep 06/09)(EScut)
- Making agarose gel
- Electrophoresis
'11/06/28(Tue)
- Digest(product of Miniprep 06/09)(EPcut)
- Electrophoresis
- Defrost and transformation(BBa_E0030,E0020,I712019,I712052,J52008)
- Making 1×TAE, agarose gel, LB medium, LB plate(amp),
'11/06/29(Wed)
- Picking up colony
- Making LB broth
'11/07/01(Fri)
- Digest(EPcut)
- Miniprep(BBa_E0030,E0020,I712019,I712052,J52008)
'11/07/05(Tue)
- Electrophoresis(product of digest 07/01)
- Digest(E0240,I712019,J52008,B0032,B0014)
- Making antibiotic stock(1000×)(Km,Cm)
'11/07/06(Wed)
- Gel extraction (pre)(BBa_I712052)
- Picking up colony(Ba_J23119,J23118,J22106,E0040?)
- Making agarose gel
'11/07/07(Thu)
- Digest(BBa_E0240,I712019,J52008,B0032,B0014)
- Defrost(BBa_E1010,K325101,K145001,I712074,R0011,C0012)
- Transformation(BBa_K145001,I712074,R0011,C0012)
- Making LB plate(Cm×10,Km×9)
'11/07/08(Fri)
- Miniprep(BBa_J23119,J23118,J22106,E0040)
'11/07/11(Mon)
- Gel extraction(E0240,I712019,J52008)
- Transformation(J23119,R0011,C0012,E1010,K325101,K145001,I712074)
'11/07/12(Tue)
- Picking up colony
'11/07/13(Wed)
- Frozen stock(J23119(18A),R0011(6G),C0012(2O),I712079(6N),K145001(2F))
- Picking up colony(E1010)
- Gel extraction(B0032,B0014)
- Digest (J23118 SPcut)
'11/07/14(Thu)
- Miniprep(E1010,J23119,K145001,I712074,R0011,C0012)
- Electrophoresis(J23118)
- Gel extraction(J23118,B0032,B0014)
- Passafe(E1010)
- Making LB+amp
'11/07/15(Fri)
- Ligation(J23118-E0240,J23118-B0032,I712019-B0014,J52008-B0014)
- Transformation(J23118-E0240,J23118-B0032,I712019-B0014,J52008-B0014)
- Dispensing Ligation Buffer
- Frozen stock(E1010)
'11/07/19(Tue)
- Digest
- Gel extraction
- Making competent cell
'11/07/20(Wed)
- Picking up colony
- Making Master plate
- Testing product of Miniprep
- Transformation (#2,#27,#28)
'11/07/21(Thu)
- Miniprep(#2-3,#2-9,#14-5,#17-5,#11,#20)
- Testing product
- Digest(#20,#2-3 SPcut #10,#11,#14-5,#17-5 XPcut)
- Making TB
'11/07/22(Fri)
- Frozen stock(#2,#2-3,#2-9,#14-5,#17-5)
- Electrophoresis(#20,#2-3 SPcut #10,#11,#14-5,#17-5 XPcut)
- Digest(#20 SPcut, #10,#11,#14-5,#17-5 XPcut)
- Transformation(#27, #28, product of Miniprep 07/20)
'11/07/25(Mon)
- Colony check(#2,#2-9)
- Gel Extraction(#20 SPcut #10,#11,#14-5,#17-5 XPcut)
- Ligation and Transformation()
- Defrost Primer(200×, 10×)
- Dispensing PCR Mix
'11/07/26(Tue)
- Picking up colony and making master plate(#20-9,#3-11,#3-14-5,#3-17-5)
- Colony PCR(#20-9,#3-11,#3-14-5,#3-17-5)
- Digest(#14-5 XPcut,Ecut, #22 SPcut, #23 XPcut)
- Ligation(#3-#14-5(Re), #3(Negative control))
- Transformation(#15,#27,#28,#30,#3-14-5(Re), #3(Negative control))
- Making reagent for TB(KOH,CaCl2,KCl,MnCl2)
'11/07/27(Wed)
- Miniprep(#1,#2,#3,#10,#22,#24,#20-9,#3-11,#3-17-5)
- Electrophoresis
'11/07/28(Thu)
- Gel extraction(#14-5 XPcut, #22 SPcut, #23 XPcut)
- Digest(#1,#2,#3,#24 SPcut, #3-11 EScut, #3-17-5 XPcut)
- Ligation(#3-#14-5)
- Making TB bugger, LB plate, 0.3% LB plate
'11/07/29(Fri)
- Cloning(lexA,cheZ)
- Colony PCR(#3-#14-5)
- Gel extraction and Agarase処理 (#1,#2,#3,#24 SPcut, #3-11 EScut, #3-17-5 XPcut, lexA,cheZ PCR product)
- Miniprep(#30)
- Digest(lexA, cheZ PCR product XP cut)
'11/08/01(Mon)
- Nanodrop(#3-11(1),#1,#3,#24,#2,#3-17-5(4)M#30)
- Digest(#3 SPcut,#5 EXcut,#9 XPcut,#30 XP cut)
- Cloning(lexA,cheZ)
- Gel extraction(#3,#9,#30,pSB1A2)
- Ligation(#2-#3-17-5,#3-11-#5,#14-#5,#3-#23,#22-#9,#3-#30)
- Transformation(#2-#3-17-5,#3-11-#5,#14-#5,#3-#23,#22-#9,#3-#30,#27(IPTG回復培養),#28)
'11/08/02(Tue)
- Colony PCR(#2-#3-17-5,#3-11-#5,#14-#5,#3-#23,#22-#9)
- Cloning(PCR)(#27,#28)
- Gel extraction
- PCR_2nd(lexA,cheZ,#27,#28)
- Digest (#30 again)
- Making Competent cell(cheZ-, Wild Type)
- Miniprep
'11/08/04(Thu)
- Electrophoresis(product of ligation #2-3-17-5(2),#3-11-5(1,2),#14-5(2),#22-9(1,2,3))
- Frozen stock(#2-3-17-5(2),#3-11-5(1,2),#14-5(2),#22-9(1,2))
- Making Gel
- Digest(#23 XPcut)
- IPTG induce(#20-9)
'11/08/05(Fri)
- Digest(#14-5(2) XPcut, #3-11-5(1) XPcut)
- Cloning(PCR)(#27, #28, lexA, cheZ)
'11/08/08(Mon)
- Defrost Frozen stock, Miniprep, Nanodrop and Electrophoresis(#5, #20, #23, #30)
- Making Gel
- Digest(#27, #28, cheZ, lexA, #3-11-5(1), #23,#30 XPcut,#6 EScut,#7 EXcut,#7,#20 SPcut)
- Defrost Frozen stock(#5,#14-5(2),#3-11-5(1))
'11/08/09(Tue)
- Miniprep, Electrophoresis and Digest(#5, #14-5(2), #3-11-5(1))
- Gel extraction and Nanodrop(#6,#27,#8,cheZ,lexA③,#3-11-5(1),#23,#7(EX),#20,#7(SP))
- Ligation(#27-psB1A2, #3-27, #20-28, #28-psB1A2, #3-29, #29-psB1A2, #31-psB1A2, #3-#23, #1-#3-11-5(1), #2-#3-11-5(1), #24-#3-11-5(1), #3-#30)
- Making LB plate
'11/08/10(Wed)
- Colony PCR(#27-pSB1A2, #3-27, #20-28, #28-psB1A2, #3-29, #29-psB1A2, #31-pSB1A2, #3-#23, #1-#3-11-5(1), #2-#3-11-5(1), #24-#3-11-5(1), #3-#30))
- Cloning(lexA, cheZ)
- Digest(#6 EScut, #7 EXcut, #1,#2,#3,#7,#20 SPcut, #23 XPcut)
- Defrost Primer
'11/08/11(Thu)
- Gel extraction(#30,#14-5(insert,vector),#5,#3-11-5,cheZ,lexA)
- Cloning(sulAp, uvrAp)
- Miniprep(#3, #20-28,#31,#24-3-11-5,#3-30)
'11/08/12(Fri)
- Colony PCR(#3-14-5(2), #6-5, #3-23)
- Cloning(nested-PCR)(uvrAp, sulAp)
- Check Electrophoresis(8/11 PCR product:#27, #28, Miniprep product:#3-14-5(2),#5)
- Digest(#5 EXcut, #14 EScut)
- Gel extraction(8/11 digest product : #5, #14-5, #3-30, cheZ, lexA, 8/11 PCR product : #27, #28 8/12 nested-PCR product : sulAp, uvrAp)
- Colony PCR (re) (#13-14-5)
- Miniprep(ligation product : #6-5, #3-23)
- Cloning(hotstart-PCR: uvrBp & cheZ)
- Digest(XPcut:#27, #28, #6-5 EScut: sulAp, uvrAp, #3-23)
'11/08/15(Mon)
- Gel extraction(from previous digest products(8/12):#27, #28, #33, #34, %, #4, #3-23)
- Digest(XPcut: cheZ, uvrBp(PCR products) EScut: #3-11-5 to obtain pSB1AK3 EScut)
- Assay of Cell lysis construction(pLuc-Lysis device) inducted by IPTG
- Making Lysis buffer, Agarose gel
- Gel extraction(from digest products XPcut: cheZ, uvrBp(PCR products) EScut: #3-11-5 to obtain pSB1AK3 EScut)
- Ligation(#3-#14-5, #24-#3-17-5, #24-#3-11-5, #20-#28, #3-#27, #3-#29, #3-23-#5, #33-pSB1AK3, #34--pSB1AK3, #35--pSB1AK3)
- Transformation(ligation products:WT, #2-9:cheZ-, #14, #17) -> 37 incubation O.N.
'11/08/16(Tue)
- Colony PCR(1:#3-#14-5, 2:#24-#3-17-5, 4:#20-#28, 5:#3-#27, 6:#3-#29, 7:#3-23-#5)
- Culture (4:#20-#28, 14:#5, 16:#14, 17:#17, 18:#2-9, #2-4, #2-3-17-5, #1, #2)
- Incubation (3:#24-#3-11-5, 8:#33-pSB1AK3, 9:#34-pSB1AK3, 10:#35-pSB1AK3)
- Colony PCR(additional)(previously incubated ligation products: 3, 8, 10)
- hotstart PCR(sulAp, uvrAp, uvrBp)
- Digest(SPcut: #3, Ecut:#3(control)) go to 37 incubation(12:30
- Ligation retry(1:#3-#14-5, 5:#3-#27, 6:#3-#29, N.C.:#3)
- Making Frozen stock(#14、#17)
- Culture test(#14 on 0.3%Agar.(km))(17:10
- Ethanol precipitation: 29.9ng/ul * 1000ul (!)
- Miniprep(Ligation products: #2-3-17-5、#14、#17、#1、#2)
- Electrophoresis(Colony PCR products: 3, 8, 10)
- Waking from frozen stock(#3, CheZ-, WT)
'11/08/17(Wed)
- Hotstart-PCR (Change conditions a little )
- from Genome
- from nested-region
- from parts
- Making Competent Cell, Sob medium
- Ethanol precipitation(WT: 40ng/ul)
- Culture from master-plate(#6-5, #20-28, #3-27, #33, #24-3-11-5 #24-3-17-5) -> 21:00 Miniprep
- Assay(IPTG induced cell lysis device
- Gel extraction(SPcut:#3)
- Culture from Frozen stock(#1, #2, #2-3-17-5)
- Electrophresis check!!!(Loading only DNA size markers)
- Miniprep(#3, #6-5, #20-28, #3-27, #33, #24-3-11-5 #24-3-17-5)
'11/08/18(Thu)
- Digest(8/17#3,#33 SPcut, #14-5 XPcut, #33 Ecut, #3-27,#3-30,#20-28 EScut, pSB1K3 EPcut)
- Hotstart-PCR (Change conditions a little )
- From Genome : uvrBp, cheZ
- From nested-region : uvrA
- Culture from 0809 master-plate ⑪ (#2-3-11-5) → Miniprep.
- Making LB plate & tube with various agarose conc. (plate: 0.5, 0.7, 0.9%×2 each, tube: 0.5,1.0,1.5,2.0,3.0%×2 each)(selection: Kanamycin)
- Gel extraction(8/17#3,#33 SPcut, #14-5 XPcut, #33 Ecut, #3-27 EScut, pSB1K3 EPcut)
- Miniprep(#1, #2, #2-3-11-5, #2-3-17-5)
- Digest(#1,#2 SPcut, #2-3-17-5 XPcut, #2-3-11-5 SPcut(check),EPcut(selection), #3-30,#20-28 EScut, 0817 Gel extracted PCR product (uvrBp) EScut, 0818PCR products (uvrAp, uvrBp, cheZ) ES&XPcut) →O.N.
- Ligation (8/17#3-#14-5, 8/17#3-8/15#29, #33-#3-11-5, 8/15#34-pSB1AK3, #3-27-#5, #20-28-pSB1AK3, #28-pSB1AK3)
- Culture for Frozen stock in eppendolf tube (O.N. in 37℃ incubator)
'11/08/19(Fri)
- ColonyPCR(from ligation products)(#3-14-5, #3-29, #33-3-11-5, #34-pSB1AK3, #3-27-5)
- Gel extraction(08/18's digest products:#1, #2, #2-3-11-5 SPcut, #2-3-17-5, cheZ XPcut, #2-3-11-5 EPcut, 8/18uvrBp, 8/17uvrBp, uvrAp EScut)
- Colony PCR retry(#3-29, #34-pSB1AK3)
- Making Frozen Stock
- Check the list of frozen stock(there were too many mistakes)
- Assay of recAp from biobrick parts using RFP construct & transilluminator
- Electrophoresis(#3, #3-27, #3-30, #6-5, #7, #20-28, #24-3-11-5, #24-3-17-5, #33)
'11/08/22(Mon)
- Making SOB medium(1000ml)
- Culture for Miniprep
- From frozen stock(#1, #2, #7, #2-3-17-5(2), #3-30)
- From master plate(#3-14-5, #33-3-11-5, #34-pSB1AK3, #3-27-5)
- From master plate retry( at 13:50 OD600=0)
- From ligation products (for mistake above)
- Miniprep, Nanodrop and Electrophoresis
- From frozen stock(#1, #2, #2-3-17-5(2), #3-30) #7 was a little bit weak
- From ligation products(#7, #3-14-5, #3-27-5, #34, #33-3-11-5)
- Digest
- From Frozen Stock (#7_EX, #30_XP, #2-3-17-5_XP, #3-30_ES)
- From Master plate(#3-14-5_XP, #34-pSB1AK3_SP, #3-27-5_XP)
- Sulvage #33 using protocol of transformation
- Culture for Frozen stock
- From Master plate(#24-3-17-5, #14-5), 700ul, in eppendolf tube
'11/08/23(Tue)
- Gel extraction
- from O.N. digest products(#7_EX, #30_XP, #2-3-17-5_XP, #3-30_ES, #3-14-5_XP, #34-pSB1AK3_SP, #3-27-5_XP)
- Frozen stock
- From O.N. culture in eppendolf tube 700ul(#24-3-17-5, #14-5)
- From O.N. culture(for Miniprep)(#34, #3-27-5, #33-3-11-5)
- Miniprep
- retry from master plate #3-14-5(278.1 ng/ul), #33-3-11-5(188.9ng/ul), #34-pSB1AK3(89.6ng/ul), #3-27-5(327.0 ng/ul) O.N. culture
- Digest (#14-5 XPcut, #3-27-5 Xcut, Pcut (check), #3-27-5 EScut (sulvage) )
- Ligation (#14-#5, #3-30-#5, #3-#30, #34-#3-11-5 #20-#28)
- #33 sulvage (isolation culture)
- Making culture(LB amp, LB)
'11/08/24(Wed)
- Gel extraction & Electrophoresis(#3-27-5_ES, #14-5_XP, #3-27-5_X, *PCR for amplification (or cloning) -> PCR clean-up system -> digest
- #27 (82.1 ng/ul, total = 30 ul) -> XP cut
- #28 (104.4 ng/ul, total = 30 ul) -> XP cut
- Making plate and amp stock →4℃
- amp 32
- km 11 (km conc. = 3/4 * that it was)
- Miniprep and Nanodrop
- #33 32.1 ng/ul <- there is probability that culture volume was not enough ->transfromation
- Luciferase assay reagents prep
- D-Luciferin Solution : 1ml*10 + 100ul*10 at -80℃
- Coelenterazine Solution : 100ul * 10 at -20℃
- Lysis buffer : 500ul * 10 at -20℃
- Ligation(#3-30-#5, #3-11-5-#34, #30-#3, #28-#20, #35-pSB1AK3, #2-3-11-5-pSB1K3, #14-5-#3 and N.C)
'11/08/25(Thu)
- Renilla luciferase assay (with #2-3-17-5 culture)
- Gel extraction (#27,#28 XPcut)
- Ligation (0824 retry & #20-#28, #3-#27)
- Transformation (Ligation products into WT competent cells & #14-5 miniprep product into WT cell (line-drawing spread) & #33 miniprep product into ΔcheZ cell (line-drawing spread))
- Digest (#14 XPcut)
- Passage(#2-3-17-5, WT, ΔcheZ, WT in eppen, ΔcheZ in eppen)
'11/08/26(Fri)
- Renilla luciferase assay (with #2-3-17-5 culture)
- Colony PCR (#33 Miniprep transformations)
- Gel extraction (#14 XPcut)
- Ligation (retry) (using today's competent cells & 0802 ΔcheZ cells)
- Cloning (cheZ)
- Digest (#5 EXcut, cheZ PCR products XPcut)
- Passage in a shaker(#2-3-17-5, WT, ΔcheZ)
- Making competent cell, LB broth
- Contamination check (0817 comp., 0802 comp., 0802 ⊿cheZ comp. spread on LB (amp) plates)
'11/08/27(Sat)
- Ligation & Transformation (0826 ①②③⑤⑧⑨) (into Nippon Gene comp.)
- Contamination check (WT FS, ΔcheZ FS)
- Waking from FS (WT, ΔcheZ)
'11/08/28(Sun)
- Making ΔcheZ comp., LB amp plates, TB
- Colony PCR & master plate preparation -> Failure!!
- Gel extraction (#29 XPcut, #5 EXcut)
- Miniprep & frozen stocks (#33, #14-5)
- Digest (#20 SPcut, #14-5 XPcut)
- Waking from Frozen stock (WT, ⊿cheZ, #20, #14-5)
'11/08/29(Mon)
- PCR purification -> digest with XP
- #27(251.3ng/ul),#28
- Miniprep
- #20(128.1ng/ul), #14-5
- Gel extraction(#14-5, #20)
- Ligation (higher I/V ratio, into cheZ comp.)(#3-#14-5(retry), #2-3-11-5 to pSB1K3 (retry), #35-pSB1AK3, (#33,#34)-#3-17-5)
'11/08/30(Tue)
- Making reagents and medium
- LB amp
- Aspartic acid solution (10mM)
- H2O2 solution (10mM)
- Gel extraction -> PCR was failed!
- #27,#28
- There was trash (from PCR) below
- Colony PCR(#3-#14-5, #2-3-11-5 to pSB1K3, #35-pSB1AK3, (#33,#34)-#3-17-5)
- PCR -> check (5uL EP -> PCR clean-up(#27,#28)
- Digest(#20_SP, pSB1AK3_EP, #3_SP)
- EP for MP check(#14-5, #20)
- Ligation(#3-#29, pSB1AK3-#2-3-11-5 on Amp/Km plate, #3 (Negative control), pSB1AK3 (Negative control) on Amp/Km plate
- Transformation(#2-9 into cheZ-) -> Line-drawing plating
- SOS stress (UV irradiation or H2O2)(#24-3-11-5, #33-3-11-5)
- culture for Miniprep from master plate(#34-3-17-5)
- Waking from frozen stock(#24-3-11-5, #33-3-11-5)
'11/08/31(Wed)
- Making LB plates(amp, km)
- Luciferin reagent preparation
- Flask A.C. for making competent cells
- Miniprep (#34-3-17-5)
- Gel extraction(#20_SP, pSB1AK3_EP, #3_SP)
- PCR -> clean-up -> XPcut(#27, #28, #14-5, #3-30)
- Waking from Frozen stock(#2-9 (WT), #24-3-17-5)
- Plating from liquid culture(#2-3-17-5)
- Ligation(#20-#3-17-5, #2-3-11-5-pSB1AK3, #3-#14, #20-#28, #3-#27, #3-#29, #35-pSB1AK3, #3-#14-5, #3-30-#5, #33-#3-17-5 & Negative control)
- Transformation
- Plasmid isolation
- #34-3-17-5(MP;1:9gradient)
- Part cloning(#36, #37)
- Contamination check(pSB1AK3(EPcut), pSB1K3(EPcut))
- Titer check(#3-17-5(Takara comp.,1:9gradient), #2-3-11-5(ΔcheZ comp.0802, 1:9gradient), #2-3-11-5(ΔcheZ comp.0830, 1:9gradient))
- Ligation products
'11/09/01(Thu)
- Colony PCR
- #34-3-17-5(MP), #3-14, #3-29, #35-pSB1AK3, #3-14-5, #37(from Part cloning)
- Gel ext.
- #27XP ,#28XP(0830); #27XP, #28XP, #3-30XP -> failed!, #14-5XP(0831)
- Making medium(SOB, LB amp plate)
- Making competent cells (WT & ΔcheZ)
- Miniprep(#3, #20)
- PCR & Column purification
- #3-14, #3-29, #3-14-5 (Recovery from colony PCR tubes!)
- Digest
- #3 -> Failed (Mixed with DMSO!), #20
- #3-14, #3-29, #3-14-5
- Ligation
- #3-27, #20-#3-17-5
- Transformation
- #36 into Nippon Gene comp.
- Ligation products into Takara comp. (including N.C.)
- Titer check(MP product #3 into 0901 WT comp., MP product #20 into 0901 ΔcheZ comp.)
- Mobility assay
- #14-5 (km resistance) WT/ΔcheZ cells
'11/09/02(Fri)
- Making medium and reagent(LB 120ml(0.125, 0.25, 0.5% Agar, M9 stock(20×), M9 200ml, Vitamins Solution (100×))
- Gel extraction -> Digest products were OK but extraction was failed.
- #20_SP, #3-14_XP, #3-14-5_XP, #3-29_XP
- Colony PCR & Master plate(#20-3-17-5, #3-27)
- Miniprep(#3, #20, #37, #3-14, #3-29, #3-14-5, #20-3-17-5, #3-27, ΔcheZ)
- Hot-start PCR & Column purification -> PCR products were OK but all purified products were lost.
- #20-3-17-5, #3-27 (Recovery from colony PCR tubes)
- #3-14, #3-14-5, #3-29 (from miniprep products)
- PCR & Column purification
- #20-3-17-5, #3-27, #3-14-5, #3-29 (from miniprep products)
- Digest
- #3_SP, #20_SP, #37_ES, #3-14_ES, #3-29_ES, #3-14-5_XP, ΔcheZ?_E/P (from MP products)
- #20-3-17-5_XP, #3-27_ES, #3-14-5_XP, #3-29_ES (from PCR products)
'11/09/03(Sat)
- Gel extraction
- Ligation & Transformation
- #20-#3-14-5, #2-#3-14-5, #3-27-#5 into Nippon Gene comp. (+ High Competent Broth 240ul)
- #3-27-#5, #37-#5 into 0902 WT comp.(+ Takara SOC broth 500ul)
- Assays
- Mobility assay (.5%, .25%, .125%)
- Asp assay
- Part Cloning (#36)
'11/09/04(Sun)
- Hot-start PCR Pre-Mix (4×) preparation (Buffer + dNTPs + THUNDER Taq)
- Colony PCR(#20-3-14-5, #3-27-5, #3-29-5, #37-5)
- Colony Diffusion Assays
- GFP Live-Imaging Assays
- Ligation & Transformation (retry)
- #2-#3-14-5, #3-27-#5, #3-29-#5, #37-#5, #20-#28 into 0901 WT comp.
- #36 into Takara comp.
- #2, #5 as Negative Controls into 0901 WT comp.
- Culture for Miniprep(#20-3-14-5, #20)
- Digest(#20)
'11/09/05(Mon)
- Colony Diffusion Assays (#2-9) WT,cheZ-
- Gel extraction(#20)
- Luciferase Assays
- Calibrations (Firefly Luciferase standards)
- Background Assay
- Dual Luciferase Assay(#20-3-14-5, #20-3-17-5)
- Colony PCR(#2-#3-14-5, #3-27-#5, #3-29-#5, #37-#5, #20-#28, #36)
- PCR -> clean-up -> Digest(#3-29-#5, #37-5)
- IPTG-inducted Cell lysis test(IPTG+, LB plate (Positive control))
- Miniprep(#20-3-14-5, #37-5, #36)
- Making M9 -> Asp -> all failed(.125%, .25% Agar)
- Culture for Miniprep(#2-3-14-5)
'11/09/06(Tue)
- Gel extraction(#37-5, #3-29-5)
- Miniprep(#2-3-14-5, #20-3-14-5, #20-3-17-5, #36, #3-29-5, #37-5)
- PCR (retry) (Hot-start & Normal PCR) -> Purification -> Digest(#3-29-5_XP, #37-5_ES)
- PCR (cloning retry)(lexA, uvrBp)
- Ligation & Transformation
- #3-27-#5, #3-30-#5, #2-3-11-5-pSB1AK3
- #5, pSB1AK3 (N.C.)
- Culture for GFP live-imaging (#2-9 WT/cheZ-) -> 4℃ storage
- Ninhydrin sol. preparation (Not dissolvable!) -> 4℃ storage
'11/09/07(Wed)
- Colony PCR(#3-27-5, #3-30-5)
- Digest(#36_SP)
- Culture from master plate(#36, #3-29-5, #37-5)
- Miniprep(#36, #3-29-5, #37-5, #3-27-5 (failed))
- PCR purification & Digest(lexA_XP, uvrBp_ES)
- Gel extraction(#3-29-5_XP, #37-5_ES, pSB1AK3_EP, #36_SP)
- PCR amplification -> Digest(#3-14-5, #3-29-5, #37-5, #3-27-5)
- Digest(#36_SP (re), #2_SP, #2-3-17-5_EX, #3-14-5_ES, #34_SP)
- Culture for Miniprep(#2, #34, #36, #3-27-5, #3-14-5, #2-3-17-5)
Ligation & Transformation
- #33-#3-14-5, #34-#3-14-5, #36-#3-14-5, #2-#3-29-5, #20-#3-29-5, #3-#37-5, #2-3-11-5-pSB1AK3, #1-#3-17-5
- #33, #34, #36, #1, #2, #20, #3, pSB1AK3 (N.C.)
- Ninhydrin stock solution preparation (10%, H2O : EtOH = 9:1)
- Asp conc. check (it took 5 min at 80C)
- Making M9 medium(.25, .125% Agar.)
'11/09/08(Thu)
- Making competent cell (WT), LB(amp) plate
- Frozen stocks(#36, #3-14-5, #3-27-5)
- Colony PCR(#33-3-14-5, #34-3-14-5, #36-3-14-5, #20-3-29-5, #2-3-11-5(psB1AK3))
- Miniprep(#2, #34, #36, #2-3-17-5, #3-27-5, #3-14-5)
- Gel extraction
- PCR product: #35_SP, #31_XP, #3-14-5_XP, #3-27-5_XP, #3-29-5_XP, #37-5_XP
- Miniprep product: #2_SP, #34_SP, #36_SP, #2-3-17-5_EX, #3-14-5_ES
- PCR amplification(#34-3-14-5, #36-3-14-5, #20-3-29-5, #20-3-14-5)
- PCR for Sequencing -> Store at -20C(#33-3-11-5, #34-3-14-5, #20-3-29-5, #37-5)
- Ligation(#35_ES-psB1AK3_ES, #31_XP-psB1A2_XP, #31_XP-psB1AK3_XP, #3-#37-5, #36-3-29-5, #3-14-5_ES-#2-3-17-5_EX, #20-#3-27-5)
- Culture for Miniprep(#34-3-17-5, #36-3-14-5, #20-3-29-5, #2-3-11-5)
- Digest
- PCR products: #34-3-14-5_ES, #36-3-14-5_ES, #20-3-14-5_ES
- pSB1C3_EP
- Asp Gradient test
'11/09/09(Fri)
- Making competent cell (WT), TB
- Colony PCR(#35-pSB1AK3 (failed), #31-pSB1AK3, #3-#37-5, #36-#3-29-5, #3-14-5-#2-3-17-5, #20-3-27-5(failed))
- PCR amplification
- From Miniprep : #3-14-5, #3-17-5
- From Colony PCR : #31, #3-37-5, #36-3-29-5, #3-14-5-2-3-17-5 (failed)
- Digest
- Miniprep product: #3-11-5_EP
- PCR-purified product: #3-14-5_EP, #3-17-5_EP, #31_EP, #3-37-5_XP, #36-3-29-5_ES
- Miniprep(#34-3-14-5, #36-3-14-5, #20-3-29-5, #2-3-11-5 (pSB1AK3))
- Gel extraction(#34-3-14-5_ES, #36-3-14-5_ES, #20-3-14-5_ES, #3-11-5_EP)
- Ligation
- #3-11-5_EP-pSB1C3_EP, #29_XP-pSB1AK3_XP
- #36-3-14-5_ES-#2-3-17-5_EX, #20_SP-#3-27-5_XP(retry)
- Comp. check
- #2-3-11-5 (pSB1AK3) into 0908 WT comp.
- #20-3-29-5 into 0909 WT comp.
- #20-3-29-5 into 0802 cheZ- comp.
- Part cloning(BBa_J04450 (IPTG-induced RFP reporter on pSB1C3))
- Culture for Miniprep(#31-pSB1AK3, #3-37-5, #36-3-29-5, #3-14-5-2-3-17-5)
- UV irradiation -> Luciferase Assay(#24-3-17-5, #34-3-17-5)
- Asp Gradient test
- CleanSEQ -> Sequencing(#33-3-11-5, #34-3-14-5, #20-3-29-5, #37-5)
'11/09/10(Sat)
- Culture plate storage (4℃)
- Miniprep(#31-pSB1AK3, #3-37-5, #36-3-29-5, #3-14-5-2-3-17-5)
- Gel extraction(#3-14-5_EP, #3-17-5_EP, #31_EP, #3-37-5_XP, #36-3-29-5_ES)
- Colony PCR(#36-3-14-5-#2-3-17-5 (Maybe Ligation was FAILURE , estimated length is 1000-1500 by electrophoresis))
- Ligation(#20_SP-#3-37-5_XP)
'11/09/11(Sun)
- Digest from Miniprep products
- #3-14-5-2-3-17-5_XP
- #36-3-14-5_SP, #20_SP -> O.N.
- Colony PCR
- #3-11-5 (pSB1C3), #29 (pSB1AK3), BBa_J04450 (pSB1C3)
- #20-#3-27-5(failed), #20-#3-37-5, #36-#3-14-5-2-3-17-5
- Gel extraction(#3-14-5-2-3-17-5_XP)
- Culture for Miniprep
- From Master plate : #20-3-37-5, #29 (pSB1AK3), BBa_J04450 (pSB1C3)
- From plate stock : #20
- From frozen stock : #2-3-17-5
- Miniprep( #36-3-14-5-2-3-17-5)
- Ligation(#20_SP-#3-14-5-2-3-17-5_XP, #1_SP-#3-14-5-2-3-17-5_XP)
- Direct Ligation
- i.PCR amplification -> Digest for >1h -> Heat kill for 20min (#20-3-37-5_XP, #36-3-14-5-2-3-17-5_ES)
- ii.Digest for >1h -> Heat kill for 20min (#36-3-14-5-2-3-17-5_SP, pSB1A3_EP, #2-3-17-5_EX)
- iii.Ligation for 30min (#20-3-14-5_ES-#2-3-17-5_EX, #36-3-14-5-2-3-17-5_SP-#20-3-37-5_XP, #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1A3_EP)
- iv.Transformation
- Making 0.125% agar LB amp plate (IPTG 1, 10, 30, 100uM)
- Cell Diffusion Assay
- #20-3-29-5 WT (IPTG 1, 10, 30, 100uM)
- #20-3-29-5 cheZ-/- (IPTG 1, 10, 30, 100uM)
- WT
- cheZ-/-
'11/9/12 (Mon)
- Colony PCR
- #20-#3-14-5-2-3-17-5
- #20-3-14-5-#2-3-17-5
- #1-#3-14-5-2-3-17-5
- #36-3-14-5-2-3-17-5-#20-3-37-5 (no colony)
- #36-3-14-5-2-3-17-5-#20-3-37-5-pSB1A3 (PCR failed?)
Note: #20-3-37-5 colonies on the master-plate are RED. Maybe #2-3-11-5...
Note: #36-3-14-5-2-3-17-5-#20-3-37-5-pSB1A3 PCR amplification doesn't work. Seems failed extension...
- PCR amplification
- #20-3-37-5 from Master plate
- #36-3-14-5-2-3-17-5 from 0911 MP product
Note: Only one #20-3-37-5 colony is NOT RED.
- Miniprep.
- #20
- #29 (pSB1AK3)
- #2-3-17-5
- #2-3-37-5
- BBa_J04450 (pSB1C3)
- Digest
- #36-3-14-5-2-3-17-5_ES (from PCR)
- #20-3-37-5_XP (from PCR)
- pSB1A3_EP
- pSB1C3_EP
- #20-3-37-5_XP (from MP) -> O/N
- J04450_EP -> O/N
- #2-9_XP -> O/N
- #20-3-29-5_ES -> O/N
- Gel extraction
- #20_SP
- #36-3-14-5_SP
- #2-3-17-5_EX (from O/N digests)
- #36-3-14-5-2-3-17-5_ES
- #20-3-37-5_XP (from today's PCR digests)
- Culture for Miniprep.
- #20-3-37-5 from 0911 master plate -> O/N
- Dual Luciferase Assay
- #20-3-14-5-2-3-17-5
- #1-3-14-5-2-3-17-5
- #36-3-14-5-2-3-17-5-20-3-37-5-pSB1A3
- Ligation -> O/N
- #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1A3_EP
- #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3_EP
- #3-27-5_XP-#20_SP
- #36-3-29-5_ES-#20-3-37-5_XP-pSB1AK3_EP
'11/9/13 (Tue)
- Colony PCR
- #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1A3_EP
- #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3_EP (failed)
- #3-27-5_XP-#20_SP (failed)
- #36-3-29-5_ES-#20-3-37-5_XP-pSB1AK3_EP
- Culture for Luciferase Assay
- #36-3-14-5-2-3-17-5-20-3-37-5(pSB1A3, pSB1C3) (IPTG 0, 1, 10, 38, 100uM)
- #20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM)
- #1-3-14-5-2-3-17-5 (IPTG 0uM)
Note: Seemes OD600 should NOT exceed 0.4~0.5, or intracellular luciferase will be saturated.
- Miniprep.
- #20-3-14-5-2-3-17-5
- #1-3-14-5-2-3-17-5
- #20-3-37-5 (from a non-red colony on the master plate)
- Gel extraction
- #20-3-37-5_XP (from non-red colony)
- #20-3-29-5_ES(failed)
- #2-9_XP, pSB1C3_EP
Note: #20-3-29-5 was not cut!
- Dual Luciferase Assay
- #1-3-14-5-2-3-17-5 (IPTG 0uM)
- #20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM)
- #36-3-14-5-2-3-17-5-20-3-37-5 (#2-3-11-5?) (IPTG 0, 1, 10, 38, 100uM)
- Sequencing preparation
- #20-3-37-5 (from a non-red colony on the master plate)
- #20-3-29-5
- #3-27-5
- #31
- Digest
- #20_E
- #3-29-5_S (from MP products) (cut check)
- Ligation
- #20_SP-#3-27-5_XP
- Transformation
- #3-14-5-2-3-17-5
- #1-3-14-5-2-3-17-5
- #20-3-14-5-2-3-17-5 (for plate stock)
- #20-3-37-5 (from a non-red colony on the master plate) (for plasmid check)
'11/09/14(Wed)
- Colony PCR
- #3-27-5_XP-#20_SP (failed)
- Culture for Luciferase Assay (from plate stocks)
- #20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM)
- #1-3-14-5-2-3-17-5 (IPTG 0uM)
- Digest
- #3-14-5, #3-17-5, #3-14-5-2-3-17-5, #1-3-14-5-2-3-17-5, #20-3-14-5-2-3-17-5 (EPcut)
- #20_SP
- Culture for Miniprep.
- From plate stock : #20, #20-3-14-5-2-3-17-5, #3-14-5-2-3-17-5, #3-17-5
- From 09/11 master plate : #3-11-5 (pSB1C3)
- Part cloning(#19: BBa_R0010)
- Ligation
- #20_SP-#3-29-5_XP
- #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3
- Making WT competent cell
- Dual Luciferase Assay
- #1-3-14-5-2-3-17-5 (IPTG 0uM)
- #20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM)
- Asp TLC Assay
- Asp Diffusion Assay (0.25% Agar)
- Fumaric Acid (Fum) Sol. preparation
- Sequencing
- ①F⑤R#20-3-37-5 (from a non-red colony on the master plate)
- ②F⑥R#20-3-29-5 (#2-3-11-5?)
- ③F⑦R#3-27-5
- ④F⑧R#31