Team:UT-Tokyo/LabNote

From 2011.igem.org

(Difference between revisions)
Line 354: Line 354:
*Miniprep(<nowiki>#</nowiki>30)
*Miniprep(<nowiki>#</nowiki>30)
*Digest(lexA, cheZ PCR product XP cut)
*Digest(lexA, cheZ PCR product XP cut)
 +
 +
=='11/08/01(Mon)==
 +
*Nanodrop(<nowiki>#</nowiki>3-11(1),<nowiki>#</nowiki>1,<nowiki>#</nowiki>3,<nowiki>#</nowiki>24,<nowiki>#</nowiki>2,<nowiki>#</nowiki>3-17-5(4)M<nowiki>#</nowiki>30)
 +
*Digest(<nowiki>#</nowiki>3 SPcut,<nowiki>#</nowiki>5 EXcut,<nowiki>#</nowiki>9 XPcut,<nowiki>#</nowiki>30 XP cut)
 +
*Cloning(lexA,cheZ)
 +
*Gel extraction(<nowiki>#</nowiki>3,<nowiki>#</nowiki>9,<nowiki>#</nowiki>30,pSB1A2)
 +
*Ligation(<nowiki>#</nowiki>2-<nowiki>#</nowiki>3-17-5,<nowiki>#</nowiki>3-11-<nowiki>#</nowiki>5,<nowiki>#</nowiki>14-<nowiki>#</nowiki>5,<nowiki>#</nowiki>3-<nowiki>#</nowiki>23,<nowiki>#</nowiki>22-<nowiki>#</nowiki>9,<nowiki>#</nowiki>3-<nowiki>#</nowiki>30)
 +
*Transformation(<nowiki>#</nowiki>2-<nowiki>#</nowiki>3-17-5,<nowiki>#</nowiki>3-11-<nowiki>#</nowiki>5,<nowiki>#</nowiki>14-<nowiki>#</nowiki>5,<nowiki>#</nowiki>3-<nowiki>#</nowiki>23,<nowiki>#</nowiki>22-<nowiki>#</nowiki>9,<nowiki>#</nowiki>3-<nowiki>#</nowiki>30,<nowiki>#</nowiki>27(IPTG回復培養),<nowiki>#</nowiki>28)
 +
 +
=='11/08/02(Tue)==
 +
*Colony PCR(<nowiki>#</nowiki>2-<nowiki>#</nowiki>3-17-5,<nowiki>#</nowiki>3-11-<nowiki>#</nowiki>5,<nowiki>#</nowiki>14-<nowiki>#</nowiki>5,<nowiki>#</nowiki>3-<nowiki>#</nowiki>23,<nowiki>#</nowiki>22-<nowiki>#</nowiki>9)
 +
*Cloning(PCR)(<nowiki>#</nowiki>27,<nowiki>#</nowiki>28)
 +
*Gel extraction
 +
*PCR_2nd(lexA,cheZ,<nowiki>#</nowiki>27,<nowiki>#</nowiki>28)
 +
*Digest (<nowiki>#</nowiki>30 again)
 +
*Making Competent cell(cheZ-, Wild Type)
 +
*Miniprep
 +
 +
=='11/08/04(Thu)==
 +
*Electrophoresis(product of ligation <nowiki>#</nowiki>2-3-17-5(2),<nowiki>#</nowiki>3-11-5(1,2),<nowiki>#</nowiki>14-5(2),<nowiki>#</nowiki>22-9(1,2,3))
 +
*Frozen stock(<nowiki>#</nowiki>2-3-17-5(2),<nowiki>#</nowiki>3-11-5(1,2),<nowiki>#</nowiki>14-5(2),<nowiki>#</nowiki>22-9(1,2))
 +
*Making Gel
 +
*Digest(<nowiki>#</nowiki>23 XPcut)
 +
*IPTG 誘導(<nowiki>#</nowiki>20-9)
 +
 +
=='11/08/05(Fri)==
 +
*Digest(<nowiki>#</nowiki>14-5(2) XPcut, <nowiki>#</nowiki>3-11-5(1) XPcut)
 +
*Cloning(PCR)(<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, lexA, cheZ)
 +
 +
=='11/08/08(Mon)==
 +
*Defrost Frozen stock, Miniprep, Nanodrop and Electrophoresis(<nowiki>#</nowiki>5, <nowiki>#</nowiki>20, <nowiki>#</nowiki>23, <nowiki>#</nowiki>30)
 +
*Making Gel
 +
*Digest(<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, cheZ, lexA, <nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>23,<nowiki>#</nowiki>30 XPcut,<nowiki>#</nowiki>6 EScut,<nowiki>#</nowiki>7 EXcut,<nowiki>#</nowiki>7,<nowiki>#</nowiki>20 SPcut)
 +
*Defrost Frozen stock(<nowiki>#</nowiki>5,<nowiki>#</nowiki>14-5(2),<nowiki>#</nowiki>3-11-5(1))
 +
 +
=='11/08/09(Tue)==
 +
*Miniprep, Electrophoresis and Digest(<nowiki>#</nowiki>5, <nowiki>#</nowiki>14-5(2), <nowiki>#</nowiki>3-11-5(1))
 +
*Gel extraction and Nanodrop(<nowiki>#</nowiki>6,<nowiki>#</nowiki>27,<nowiki>#</nowiki>8,cheZ,lexA③,<nowiki>#</nowiki>3-11-5(1),<nowiki>#</nowiki>23,<nowiki>#</nowiki>7(EX),<nowiki>#</nowiki>20,<nowiki>#</nowiki>7(SP))
 +
*Ligation(<nowiki>#</nowiki>27-psB1A2, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>28-psB1A2, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>29-psB1A2, <nowiki>#</nowiki>31-psB1A2, <nowiki>#</nowiki>3-<nowiki>#</nowiki>23, <nowiki>#</nowiki>1-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>2-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>24-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>3-<nowiki>#</nowiki>30)
 +
*Making LB plate
 +
 +
=='11/08/10(Wed)==
 +
*Colony PCR(<nowiki>#</nowiki>27-pSB1A2, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>28-psB1A2, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>29-psB1A2, <nowiki>#</nowiki>31-pSB1A2, <nowiki>#</nowiki>3-<nowiki>#</nowiki>23, <nowiki>#</nowiki>1-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>2-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>24-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>3-<nowiki>#</nowiki>30）)
 +
*Cloning(lexA, cheZ)
 +
*Digest(<nowiki>#</nowiki>6 EScut, <nowiki>#</nowiki>7 EXcut, <nowiki>#</nowiki>1,<nowiki>#</nowiki>2,<nowiki>#</nowiki>3,<nowiki>#</nowiki>7,<nowiki>#</nowiki>20 SPcut, <nowiki>#</nowiki>23 XPcut）
 +
*Defrost Primer
 +
 +
=='11/08/11(Thu)==
 +
*Gel extraction(<nowiki>#</nowiki>30,<nowiki>#</nowiki>14-5(insert,vector),<nowiki>#</nowiki>5,<nowiki>#</nowiki>3-11-5,cheZ,lexA)
 +
*Cloning(sulAp, uvrAp)
 +
*Miniprep(<nowiki>#</nowiki>3, <nowiki>#</nowiki>20-28,<nowiki>#</nowiki>31,<nowiki>#</nowiki>24-3-11-5,<nowiki>#</nowiki>3-30)
 +
 +
=='11/08/12(Fri)==
 +
*Colony PCR(<nowiki>#</nowiki>3-14-5(2), <nowiki>#</nowiki>6-5, <nowiki>#</nowiki>3-23)
 +
*Cloning（nested-PCR）(uvrAp, sulAp)
 +
*Check Electrophoresis（8/11 PCR product：<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, Miniprep product：<nowiki>#</nowiki>3-14-5(2),<nowiki>#</nowiki>5）
 +
*Digest（<nowiki>#</nowiki>5 EXcut, <nowiki>#</nowiki>14 EScut）
 +
*Gel extraction(8/11 digest product : <nowiki>#</nowiki>5, <nowiki>#</nowiki>14-5, <nowiki>#</nowiki>3-30, cheZ, lexA, 8/11 PCR product : <nowiki>#</nowiki>27, <nowiki>#</nowiki>28 8/12 nested-PCR product : sulAp, uvrAp)
 +
*Colony PCR (re) (<nowiki>#</nowiki>13-14-5)
 +
*Miniprep(ligation product : <nowiki>#</nowiki>6-5, <nowiki>#</nowiki>3-23)
 +
*Cloning(hotstart-PCR: uvrBp & cheZ)
 +
*Digest(XPcut:<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, <nowiki>#</nowiki>6-5 EScut: sulAp, uvrAp, <nowiki>#</nowiki>3-23)
 +
 +
=='11/08/15(Mon)==
 +
*Gel extraction(from previous digest products(8/12):<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, <nowiki>#</nowiki>33, <nowiki>#</nowiki>34, %, <nowiki>#</nowiki>4, <nowiki>#</nowiki>3-23)
 +
*Digest(XPcut: cheZ, uvrBp(PCR products) EScut: <nowiki>#</nowiki>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: <nowiki>#</nowiki>3-11-5 to obtain pSB1AK3 EScut)
 +
*Ligation(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>24-<nowiki>#</nowiki>3-17-5, <nowiki>#</nowiki>24-<nowiki>#</nowiki>3-11-5, <nowiki>#</nowiki>20-<nowiki>#</nowiki>28, <nowiki>#</nowiki>3-<nowiki>#</nowiki>27, <nowiki>#</nowiki>3-<nowiki>#</nowiki>29, <nowiki>#</nowiki>3-23-<nowiki>#</nowiki>5, <nowiki>#</nowiki>33-pSB1AK3, <nowiki>#</nowiki>34--pSB1AK3, <nowiki>#</nowiki>35--pSB1AK3)
 +
*Transformation(ligation products:WT, <nowiki>#</nowiki>2-9:cheZ-, <nowiki>#</nowiki>14, <nowiki>#</nowiki>17) -> 37 incubation O.N.
 +
 +
=='11/08/16(Tue)==
 +
*Colony PCR(1:<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, 2:<nowiki>#</nowiki>24-<nowiki>#</nowiki>3-17-5, 4:<nowiki>#</nowiki>20-<nowiki>#</nowiki>28, 5:<nowiki>#</nowiki>3-<nowiki>#</nowiki>27, 6:<nowiki>#</nowiki>3-<nowiki>#</nowiki>29, 7:<nowiki>#</nowiki>3-23-<nowiki>#</nowiki>5)
 +
*Culture (4:<nowiki>#</nowiki>20-<nowiki>#</nowiki>28, 14:<nowiki>#</nowiki>5, 16:<nowiki>#</nowiki>14, 17:<nowiki>#</nowiki>17, 18:<nowiki>#</nowiki>2-9, <nowiki>#</nowiki>2-4, <nowiki>#</nowiki>2-3-17-5, <nowiki>#</nowiki>1, <nowiki>#</nowiki>2)
 +
*Incubation (3:<nowiki>#</nowiki>24-<nowiki>#</nowiki>3-11-5, 8:<nowiki>#</nowiki>33-pSB1AK3, 9:<nowiki>#</nowiki>34-pSB1AK3, 10:<nowiki>#</nowiki>35-pSB1AK3)
 +
*Colony PCR(additional)(previously incubated ligation products: 3, 8, 10)
 +
*hotstart PCR(sulAp, uvrAp, uvrBp)
 +
*Digest(SPcut: <nowiki>#</nowiki>3, Ecut:<nowiki>#</nowiki>3(control)) go to 37 incubation(12:30
 +
*Ligation retry(1:<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, 5:<nowiki>#</nowiki>3-<nowiki>#</nowiki>27, 6:<nowiki>#</nowiki>3-<nowiki>#</nowiki>29, N.C.:<nowiki>#</nowiki>3)
 +
*Making Frozen stock(<nowiki>#</nowiki>14、<nowiki>#</nowiki>17)
 +
*Culture test(<nowiki>#</nowiki>14 on 0.3%Agar.(km))(17:10
 +
*Ethanol precipitation: 29.9ng/ul * 1000ul (!)
 +
*Miniprep(Ligation products: <nowiki>#</nowiki>2-3-17-5、<nowiki>#</nowiki>14、<nowiki>#</nowiki>17、<nowiki>#</nowiki>1、<nowiki>#</nowiki>2)
 +
*Electrophoresis(Colony PCR products: 3, 8, 10)
 +
*Waking from frozen stock(<nowiki>#</nowiki>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(<nowiki>#</nowiki>6-5, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>33, <nowiki>#</nowiki>24-3-11-5 <nowiki>#</nowiki>24-3-17-5) -> 21:00 Miniprep
 +
*Assay(IPTG induced cell lysis device
 +
*Gel extraction(SPcut:<nowiki>#</nowiki>3)
 +
*Culture from Frozen stock(<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>2-3-17-5)
 +
*Electrophresis check!!!(Loading only DNA size markers)
 +
*Miniprep(<nowiki>#</nowiki>3, <nowiki>#</nowiki>6-5, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>33, <nowiki>#</nowiki>24-3-11-5 <nowiki>#</nowiki>24-3-17-5)
 +
 +
=='11/08/18(Thu)==
 +
*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 )
 +
◦ uvrBp, cheZ from Genome
 +
◦ uvrA from nested-region
 +
*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)
 +
*Gel extraction(8/17<nowiki>#</nowiki>3,<nowiki>#</nowiki>33 SPcut, <nowiki>#</nowiki>14-5 XPcut, <nowiki>#</nowiki>33 Ecut, <nowiki>#</nowiki>3-27 EScut, pSB1K3 EPcut)
 +
*Miniprep(<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>2-3-11-5, <nowiki>#</nowiki>2-3-17-5)
 +
*Digest(<nowiki>#</nowiki>1,<nowiki>#</nowiki>2 SPcut, <nowiki>#</nowiki>2-3-17-5 XPcut, <nowiki>#</nowiki>2-3-11-5 SPcut(check),EPcut(selection), <nowiki>#</nowiki>3-30,<nowiki>#</nowiki>20-28 EScut, 0817 Gel extracted PCR product (uvrBp) EScut, 0818PCR products (uvrAp, uvrBp, cheZ) ES&XPcut) →O.N.
 +
*Ligation (8/17<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, 8/17<nowiki>#</nowiki>3-8/15<nowiki>#</nowiki>29, <nowiki>#</nowiki>33-<nowiki>#</nowiki>3-11-5, 8/15<nowiki>#</nowiki>34-pSB1AK3, <nowiki>#</nowiki>3-27-<nowiki>#</nowiki>5, <nowiki>#</nowiki>20-28-pSB1AK3, <nowiki>#</nowiki>28-pSB1AK3)
 +
*Culture for Frozen stock in eppendolf tube (O.N. in 37℃ incubator)
 +
 +
=='11/08/19(Fri)==
 +
*ColonyPCR(from ligation products)(<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>33-3-11-5, <nowiki>#</nowiki>34-pSB1AK3, <nowiki>#</nowiki>3-27-5)
 +
*Gel extraction(08/18's digest products:<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>2-3-11-5 SPcut, <nowiki>#</nowiki>2-3-17-5, cheZ XPcut, <nowiki>#</nowiki>2-3-11-5 EPcut, 8/18uvrBp, 8/17uvrBp, uvrAp EScut)
 +
*Colony PCR retry(<nowiki>#</nowiki>3-29, <nowiki>#</nowiki>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(<nowiki>#</nowiki>3, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>3-30, <nowiki>#</nowiki>6-5, <nowiki>#</nowiki>7, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>24-3-11-5, <nowiki>#</nowiki>24-3-17-5, <nowiki>#</nowiki>33)
 +
 +
=='11/08/22(Mon)==
 +
*Making SOB medium(1000ml)
 +
*Culture for Miniprep
 +
**From frozen stock(<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>7, <nowiki>#</nowiki>2-3-17-5(2), <nowiki>#</nowiki>3-30)
 +
**From master plate(<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>33-3-11-5, <nowiki>#</nowiki>34-pSB1AK3, <nowiki>#</nowiki>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(<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>2-3-17-5(2), <nowiki>#</nowiki>3-30) <nowiki>#</nowiki>7 was a little bit weak
 +
**From ligation products(<nowiki>#</nowiki>7, <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-27-5, <nowiki>#</nowiki>34, <nowiki>#</nowiki>33-3-11-5)
 +
*Digest
 +
**From Frozen Stock (<nowiki>#</nowiki>7_EX, <nowiki>#</nowiki>30_XP, <nowiki>#</nowiki>2-3-17-5_XP, <nowiki>#</nowiki>3-30_ES)
 +
**From Master plate(<nowiki>#</nowiki>3-14-5_XP, <nowiki>#</nowiki>34-pSB1AK3_SP, <nowiki>#</nowiki>3-27-5_XP)
 +
*Sulvage <nowiki>#</nowiki>33 using protocol of transformation
 +
*Culture for Frozen stock
 +
**From Master plate(<nowiki>#</nowiki>24-3-17-5, <nowiki>#</nowiki>14-5), 700ul, in eppendolf tube
 +
 +
=='11/08/23(Tue)==
 +
*Gel extraction
 +
**from O.N. digest products(<nowiki>#</nowiki>7_EX, <nowiki>#</nowiki>30_XP, <nowiki>#</nowiki>2-3-17-5_XP, <nowiki>#</nowiki>3-30_ES, <nowiki>#</nowiki>3-14-5_XP, <nowiki>#</nowiki>34-pSB1AK3_SP, <nowiki>#</nowiki>3-27-5_XP)
 +
*Frozen stock
 +
**From O.N. culture in eppendolf tube 700ul(<nowiki>#</nowiki>24-3-17-5, <nowiki>#</nowiki>14-5)
 +
**From O.N. culture(for Miniprep)(<nowiki>#</nowiki>34, <nowiki>#</nowiki>3-27-5, <nowiki>#</nowiki>33-3-11-5)
 +
*Miniprep
 +
**retry from master plate <nowiki>#</nowiki>3-14-5(278.1 ng/ul), <nowiki>#</nowiki>33-3-11-5(188.9ng/ul), <nowiki>#</nowiki>34-pSB1AK3(89.6ng/ul), <nowiki>#</nowiki>3-27-5(327.0 ng/ul) O.N. culture
 +
*Digest (<nowiki>#</nowiki>14-5 XPcut, <nowiki>#</nowiki>3-27-5 Xcut, Pcut (check), <nowiki>#</nowiki>3-27-5 EScut (sulvage) )
 +
*Ligation (<nowiki>#</nowiki>14-<nowiki>#</nowiki>5, <nowiki>#</nowiki>3-30-<nowiki>#</nowiki>5, <nowiki>#</nowiki>3-<nowiki>#</nowiki>30, <nowiki>#</nowiki>34-<nowiki>#</nowiki>3-11-5 <nowiki>#</nowiki>20-<nowiki>#</nowiki>28)
 +
*<nowiki>#</nowiki>33 sulvage (isolation culture)
 +
*Making culture(LB amp, LB)
 +
 +
=='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 O.N.
 +
**<nowiki>#</nowiki>27 (82.1 ng/ul, total = 30 ul) -> XP cut
 +
**<nowiki>#</nowiki>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
 +
**<nowiki>#</nowiki>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(<nowiki>#</nowiki>3-30-<nowiki>#</nowiki>5, <nowiki>#</nowiki>3-11-5-<nowiki>#</nowiki>34, <nowiki>#</nowiki>30-<nowiki>#</nowiki>3, <nowiki>#</nowiki>28-<nowiki>#</nowiki>20, <nowiki>#</nowiki>35-pSB1AK3, <nowiki>#</nowiki>2-3-11-5-pSB1K3, <nowiki>#</nowiki>14-5-<nowiki>#</nowiki>3 and N.C)
 +
 +
=='11/08/25(Thu)==
 +
*Renilla luciferase assay (with <nowiki>#</nowiki>2-3-17-5 culture)
 +
*Gel extraction (<nowiki>#</nowiki>27,<nowiki>#</nowiki>28 XPcut)
 +
*Ligation (0824 retry & <nowiki>#</nowiki>20-<nowiki>#</nowiki>28, <nowiki>#</nowiki>3-<nowiki>#</nowiki>27)
 +
*Transformation (Ligation products into WT competent cells & <nowiki>#</nowiki>14-5 miniprep product into WT cell (line-drawing spread) & <nowiki>#</nowiki>33 miniprep product into ΔcheZ cell (line-drawing spread))
 +
*Digest (<nowiki>#</nowiki>14 XPcut)
 +
*Passage（<nowiki>#</nowiki>2-3-17-5, WT, ΔcheZ, WT in eppen, ΔcheZ in eppen)
 +
 +
=='11/08/26(Fri)==
 +
*Renilla luciferase assay (with <nowiki>#</nowiki>2-3-17-5 culture)
 +
*Colony PCR (<nowiki>#</nowiki>33 Miniprep transformations)
 +
*Gel extraction (<nowiki>#</nowiki>14 XPcut)
 +
*Ligation (retry) (using today's competent cells & 0802 ⊿cheZ cells)
 +
*Cloning (cheZ)
 +
*Digest (<nowiki>#</nowiki>5 EXcut, cheZ PCR products XPcut)
 +
*Passage in a shaker（<nowiki>#</nowiki>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 ⊿cheZ comp., LB amp plates, TB
 +
*Colony PCR & master plate preparation -> Failure!!
 +
*Gel extraction (<nowiki>#</nowiki>29 XPcut, <nowiki>#</nowiki>5 EXcut)
 +
*Miniprep & frozen stocks (<nowiki>#</nowiki>33, <nowiki>#</nowiki>14-5)
 +
*Digest (<nowiki>#</nowiki>20 SPcut, <nowiki>#</nowiki>14-5 XPcut)
 +
*Waking from Frozen stock (WT, ⊿cheZ, <nowiki>#</nowiki>20, <nowiki>#</nowiki>14-5)
 +
 +
=='11/08/29(Mon)==
 +
*PCR purification -> digest with XP
 +
**<nowiki>#</nowiki>27(251.3ng/ul),<nowiki>#</nowiki>28(199.7ng/ul)
 +
*Miniprep
 +
**<nowiki>#</nowiki>20(128.1ng/ul), <nowiki>#</nowiki>14-5(48.3ng/ul)
 +
*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)
 +
 +
=='11/08/30(Tue)==
 +
*Making reagents and medium
 +
**LB amp
 +
**Aspartic acid solution (10mM)
 +
**H2O2 solution (10mM)
 +
*Gel extraction -> PCR was failed!
 +
**<nowiki>#</nowiki>27,<nowiki>#</nowiki>28
 +
*There was trash (from PCR) below
 +
*Colony PCR(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>2-3-11-5 to pSB1K3, <nowiki>#</nowiki>35-pSB1AK3, (<nowiki>#</nowiki>33,<nowiki>#</nowiki>34)-<nowiki>#</nowiki>3-17-5)
 +
*PCR -> check (5uL EP -> PCR clean-up(<nowiki>#</nowiki>27,<nowiki>#</nowiki>28)
 +
*Digest(<nowiki>#</nowiki>20_SP, pSB1AK3_EP, <nowiki>#</nowiki>3_SP)
 +
*EP for MP check(<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>20)
 +
*Ligation(<nowiki>#</nowiki>3-<nowiki>#</nowiki>29, pSB1AK3-<nowiki>#</nowiki>2-3-11-5 on Amp/Km plate, <nowiki>#</nowiki>3 (Negative control), pSB1AK3 (Negative control) on Amp/Km plate
 +
*Transformation(<nowiki>#</nowiki>2-9 into cheZ-) -> Line-drawing plating
 +
*SOS stress (UV irradiation or H2O2)(<nowiki>#</nowiki>24-3-11-5, <nowiki>#</nowiki>33-3-11-5)
 +
*culture for Miniprep from master plate(<nowiki>#</nowiki>34-3-17-5)
 +
*Waking from frozen stock(<nowiki>#</nowiki>24-3-11-5, <nowiki>#</nowiki>33-3-11-5)
 +
 +
=='11/08/31(Wed)==
 +
*Making LB plates(amp, km)
 +
*Luciferin reagent preparation
 +
*Flask A.C. for making competent cells
 +
*Miniprep (<nowiki>#</nowiki>34-3-17-5)
 +
*Gel extraction(<nowiki>#</nowiki>20_SP, pSB1AK3_EP, <nowiki>#</nowiki>3_SP)
 +
*PCR -> clean-up -> XPcut(<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, <nowiki>#</nowiki>14-5, <nowiki>#</nowiki>3-30)
 +
*Waking from Frozen stock(<nowiki>#</nowiki>2-9 (WT), <nowiki>#</nowiki>24-3-17-5)
 +
*Plating from liquid culture(<nowiki>#</nowiki>2-3-17-5)
 +
*Ligation(<nowiki>#</nowiki>20-<nowiki>#</nowiki>3-17-5, <nowiki>#</nowiki>2-3-11-5-pSB1AK3, <nowiki>#</nowiki>3-<nowiki>#</nowiki>14, <nowiki>#</nowiki>20-<nowiki>#</nowiki>28, <nowiki>#</nowiki>3-<nowiki>#</nowiki>27, <nowiki>#</nowiki>3-<nowiki>#</nowiki>29, <nowiki>#</nowiki>35-pSB1AK3, <nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>3-30-<nowiki>#</nowiki>5, <nowiki>#</nowiki>33-<nowiki>#</nowiki>3-17-5 & Negative control)
 +
*Transformation
 +
**Plasmid isolation
 +
**<nowiki>#</nowiki>34-3-17-5(MP;1:9gradient)
 +
**Part cloning(<nowiki>#</nowiki>36, <nowiki>#</nowiki>37)
 +
**Contamination check(pSB1AK3(EPcut), pSB1K3(EPcut))
 +
**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
=='11/9/12 (Mon)==
=='11/9/12 (Mon)==

Revision as of 01:47, 15 September 2011

Parts List

Table 1. List of iGEM parts
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

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'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 誘導(#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 )

◦ uvrBp, cheZ from Genome ◦ uvrA from nested-region

  • 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 O.N.
    • #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 ⊿cheZ 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 ⊿cheZ 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(199.7ng/ul)
  • Miniprep
    • #20(128.1ng/ul), #14-5(48.3ng/ul)
  • 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

'11/9/12 (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 ext.
    • #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 -> O/N
    • #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)

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