Team:UniSalento Lecce/Notebook

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<br />

<h2 style="text-align: center">Lab Notebook</h2>

<p>

Here you can follow our experiments and results since the beginning of our work. Click on a day in the calendar to find out how we spent those neverending days in the lab! (We thank Emanuele for keeping an up-to-date journal!)<br><br><br>

</p>

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<!-- ******************************************* Start Calendar ************************************ -->

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{

"date":"1380873600000", /*04 Ottobre*/     

"type":"demo",

"title":"Wiki freeze.",

"description":" ",

"url":"#"

},

{

"date":"1380787200000", /*03 Ottobre*/     

"type":"demo",

"title":"Play Decide partecipation.",

"description":" ",

"url":"#"

},

{

"date":"1380268800000", /*27 settembre*/     

"type":"demo",

"title":"PLATES RESULTS: no growth.<br>Boiling prep of 10, 15, 15pLac, nikR + control gel (loading order: L; 10 A; 10 B; 10 C; 10 D; nikR 1; nikR 2; 15 A; 15 B; 15 C; 15 D; 15 E; 15pLac A; 15pLac B; 15 pLac C; 15pLac D; 15 pLac E; CTRL PSB1C3).<br>RESULTS: 15pLac 5 is a good sample, so proceed with digestion and PCR. For the other samples we need new inocula. BL21 inocula and growth (2h) fluorescent decay assay with constructs 1+4 and 1+5 (CTRL, + Ni, + IPTG, + Ni + IPTG).<br>RESULTS: published on the Register (Team Parts).",

"description":" ",

"url":"#"

},

{

"date":"1380182400000", /*26 settembre*/     

"type":"demo",

"title":"PLATES RESULTS: No growth observed on NikR and 1+3 plates.<br>Remake the cloning for these samples.<br>Inocula for the other plates grown and preinocula for the two BL21 samples.",

"description":" ",

"url":"#"

},

{

"date":"1380096000000", /*25 settembre*/     

"type":"demo",

"title":"PLATES RESULTS: no growth observed.<br>Control digestion (single and double) and PCR of: 12.3, 12.4, 12.5, 1+5 A, 1+5 B, 1+5 D, 1+5 E, nikR 1, nikR 2.<br>Control gel (loading order digestions: L; nikR 1; nikR 2; 12.3; 12.4; 12.5; 1+5 A (Eco + Pst); 1+5 A; 1+5 B; 1+5 D; 1+5 E (Eco); loading order PCR: 12.3; 12.4; 12.5; 1+5 A; 1+5 B; 1+5 D; 1+5 E; nikR 1; nikR 2).<br>Rebuild the constructs 1+3 and nikR.<br>Digestion and ligation: 6 Amp C ES + 1 XP + PSB1C3 EP; 14.3 XP + B0034 ES + PSB1K3 EP; 14.3 XP + J04500 ES + PSB1A3 EP; nikR EP + PSB1C3 EP; 1 ES + 3 XP + PSB1C3 EP.<br>Transformation of DH5α cells and plate of 10, 15, 15-pLac, NikR and 1+3 in PSB1C3, BL21 1+5 and BL21 1+4 in LB agar + IPTG 1mM + related antibiotics.",

"description":" ",

"url":"#"

},

{

"date":"1380009600000", /*24 settembre*/     

"type":"demo",

"title":"Ligations of the following constructs: 6 Amp C + 1 in PSB1C3 (construct n.10), 14.3 + B0034 in PSB1K3 (construct n.15), 14.3 + pLac-RBS (J04500) in PSB1A3 (pLac-RBS + construct n.15) 6 Amp C digested with Eco and Spe, 1 with Xba and Pst; 14.3 digested with Xba and Pst, B0034 with Eco and Spe; pLac-RBS (J04500) with Eco and Spe, 14.3 with Xba and Pst.<br>Transformation and plate on LB agar + IPTG 1mM + related antibiotics.<br>Boiling prep of the yesterday’s inocula + control gel (loading order: M; nikR 1÷5; 12 1÷5; 1+5 A÷E; 1+3 A÷E).",

"description":" ",

"url":"#"

},

{

"date":"1379923200000", /*23 settembre*/     

"type":"demo",

"title":"Digestion and PCR to control the last boiling preps (2A, 3.1, 4.2, 14.3, pUreA1, Hpn1, 1+4 A, 1+4 C, 12.2, 1+5 A, 6 Amp C, 6 Kan 2ul C, 6 Kan 5ul A, 4.3, nikR A) + control gel (loading order digestions: L; 2A; 3.1; 4.3; 4.2; 12.2; 14.3; pUreA 1; Hpn 1; NikR A; 6 Amp C; 6 Kan 2ul C; 6 Kan 5ul A; 1+4 C; 1+5 A; 1+4 A. loading order PCR: L; 2A; 3.1; 4.2; 14.3; pUreA 1; Hpn 1; 1+4 A; 1+4 C; 12.2; 1+5 A; 6 AmpC; 6 Kan 2 ul C; 6 Kan 5 ul A; 4.3; nikR A).<br>RESULTS: next inocula: 12, nikR in C; 1+5; 1+3 (5 tubes per sample). ",

"description":" ",

"url":"#"

},

{

"date":"1379836800000", /*22 settembre*/     

"type":"demo",

"title":"Boiling prep of the yesterday’s inocula + control gel (loading order: L; 2A,B; 1+3 A,B,C; 1+4 A,B,C; 1+5 A,B,C; M; L; nikR A,B; 6 in AMP A,B,C; 6.2 in KAN A,B,C; 6.5 in KAN A,B,C; M).",

"description":" ",

"url":"#"

},

{

"date":"1379750400000", /*21 settembre*/     

"type":"demo",

"title":"Preparation of new LB + CAF plates.<br>Boiling prep of the yesterday’s inocula + control gel (loading order: L; 2.1; 2.2; 3.1; 3.2; 4.1; 4.2; 4.3; 4.4; 12.1; 12.2; 12.3; 12.4; M; 14.1; 14.2; 14.3; 14.4; pUreA 1÷4; Hpn 1÷4; M). (M: PSB1C3 [0,8 ug/ul]).<br>RESULTS: good boiling profile, to be confirmed by digestion.<br>Inocula of the yesterday’s plates (nikR in CAF A,B; 2 in CAF A,B; 1+3 in CAF A,B,C; 1+4 in KAN A,B,C; 1+5 in KAN A,B,C; 6 in AMP A,B,C; 6 (2 ul ligated) in KAN A,B,C; 6 (5 ul ligated) in KAN A,B,C; + 2 CTRL) (24 tubes tot.).",

"description":" ",

"url":"#"

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{

"date":"1379664000000", /*20 settembre*/     

"type":"demo",

"title":"Preparation of LB + CAF plates.<br>PLATES RESULTS: growth observed on: constructs n.2,3,4, 6, 12, 14, pUreA, Hpn inocula in LB broth + related antibiotics. Colonies on construct n.6 plate are red, so we have to rebuild the plasmid from the digestion of the single construct (the same for nikR, pint, 1, 5, pexbB, 13, pnik, 2, 3).<br>Double digestion of: PSB1A3 (EP), PSB1K3 (EP), construct n.1 (ES), constructs n. 3, 4, 5 (XP) (some parts had already been digested yesterday).<br>Ligation of: construct n.6 in PSB1A3, construct n.6 in PSB1K3, nikR in PSB1C3, pint in PSB1C3, 1 in PSB1C3, 5 in PSB1C3, pexbB in PSB1C3, 13 in PSB1C3, pnik in PSB1C3, 2 in PSB1C3, 3 in PSB1C3, 1+3 in PSB1C3, 1+4 in PSB1K3, 1+5 in PSB1K3.<br>Transformation and plate on LB agar + IPTG 1 mM + related antibiotics.",

"description":" ",

"url":"#"

},

{

"date":"1379577600000", /*19 settembre*/     

"type":"demo",

"title":"Quantification on gel of the following samples: K805016 boiling; I0462 (1); I0462 (2); I0462 (3); PCR pnik; PCR int. tot; PCR pexbB.<br>RESULTS: respective concentrations: 200 ng/ul; 50 ng/ul; 30 ng/ul; 50 ng/ul; 80 ng/ul; 80 ng/ul; 100 ng/ul.<br>Preparation of LB + KAN, CAF and AMP plates.<br>Double digestion of the following parts or constructs: PSB1A3 [0,8 ug/ul], pUreA [250 ng/ul], K805016 [200 ng/ul], PSB1C3 [0,8ug/ul], I0462 [50 ng/ul], Hpn [250 ng/ul], B0015 [0,6 ug/ul], nikR [10 ng/ul], mini 1 [300 ng/ul], mini 2 [100 ng/ul], mini 3 [100 ng/ul], mini 4 [200 ng/ul], mini 5 [100 ng/ul], mini 13 [100 ng/ul], pint [100 ng/ul], pexbB [80 ng/ul], pnik [80 ng/ul] → PSB1A3 with Eco and Pst, pUreA with Eco and Spe, pUreA with Eco and Pst, K805016 with Xba and Pst, PSB1C3 with Eco and Pst, I0462 with Xba and Pst, Hpn with Eco and Spe, Hpn with Eco and Pst, B0015 with Xba and Pst, nikR with Eco and Pst, mini 1 with Eco and Pst, mini 1 with Xba and Pst, mini 1 with Eco and Spe, mini 2 with Eco and Pst, mini 3 with Eco and Pst, mini 4 with Eco and Pst, mini 5 with Eco and Pst, mini 13 with Eco and Pst, pint with Eco and Pst, pexbB with Eco and Pst, pnik with Eco and Pst. Ligation (Takara method): schemes: PSB1A3 EP + pUreA ES + K805016 XP (construct n.6); PSB1C3 EP + pUreA ES + I0462 XP (construct n.12); PSB1C3 EP + Hpn ES + B0015 XP (construct n.14); Hpn EP + PSB1C3 EP; pUreA EP + PSB1C3 EP; nikR EP + PSB1C3 EP; 1 EP + PSB1C3 EP; 2 EP + PSB1C3 EP; 3 EP + PSB1C3 EP; 4 EP + PSB1C3 EP; 5 EP + PSB1C3 EP; 13 EP + PSB1C3 EP; pexbB EP + PSB1C3 EP; pnik EP + PSB1C3 EP; pint EP + PSB1C3 EP. Transformation and plate on LB agar + IPTG 1 mM + related antibiotics.",

"description":" ",

"url":"#"

},

{

"date":"1379491200000", /*18 settembre*/     

"type":"demo",

"title":"Boiling prep of the construct n.6 + control gel (loading order: M; 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; PSB1K3 [0,2 ug/ul]).<br>RESULTS: negative boiling profile.<br>New ligation of the constructs 14 and 12 in PSB1C3, transformation of DH5α cells and plate on LB + CAF + IPTG 1 mM (1 plate per construct).",

"description":" ",

"url":"#"

},

{

"date":"1379404800000", /*17 settembre*/     

"type":"demo",

"title":"Miniprep of the construct n.1 and boiling preps of the constructs 14 and 12.<br>Control gel (loading order: M; 14.1; 14.2; 14.3; 14.4; 14.5; 14.6; 14.8; 14.9; 14.10; 14.11; 14.12; 12.1; 12.2; 12.3; 12.4; 12.5; 12.6; 12.8; 12.9; 12.10; 12.11; 12.12; 1 mini).<br>RESULTS: low quality of the gel, all the samples of the constructs 14 and 12 checked in a new gel with PSB1C3 control; quantification on gel: 1 mini: 300 ng/ul.<br>New control gel (same loading order + PSB1C3).<br>RESULTS: no positive results, rebuild the constructs 14 and 12.<br>Inocula of the construct n.6 samples in LB + KAN (10 tubes + CTRL).",

"description":" ",

"url":"#"

},

{

"date":"1379318400000", /*16 settembre*/     

"type":"demo",

"title":"PLATES RESULTS: 12 NEW ok, 12 OLD no results.<br>Ligation (Takara method) of a new construct n.6 (pUreA ES + K805016 XP) in PSB1K3 (all the parts were already digested).<br>Transformation and plate on LB agar + KAN + IPTG 1 mM (2 plates).<br>Inocula of: 1F (LB + AMP) for miniprep, 14 NEW and 12 NEW (LB + CAF) for boiling prep (1F: 1 tube; 14 NEW: 12 tubes; 12 NEW: 12 tubes).",

"description":" ",

"url":"#"

},

{

"date":"1379059200000", /*13 settembre*/     

"type":"demo",

"title":"Double digestion (with Eco and Spe; NEB protocol) of the 5 sample (construct n.6).<br>Control gel (loading order: M; boiling 6; 6 digested).<br>RESULTS: no positive result.<br>Transformation of DH5α cells with the construct n.12 (already built: called “12 OLD”) and new construct n.12 (just ligated: called “12 NEW”) and plate on LB agar + CAF + IPTG 1 mM. AGE to control PSB1A3 integrity (2 samples checked, “AMP” and “A”).<br>RESULTS: “AMP” is a good sample (AMP: 0,8 ug/ul; A: 0,2 ug/ul).",

"description":" ",

"url":"#"

},

{

"date":"1378972800000", /*12 settembre*/     

"type":"demo",

"title":"Boiling prep of the construct n.6 (12 samples).<br>Control gel (loading order: M; 1÷12).<br>RESULTS: probable positive samples: 2, 3, 5, 7, 9 (5 maybe the best, 300 ng/ul).<br>Ligation (Takara method) of the constructs 12 and 14 (parts already digested) in PSB1C3, transformation and plate on LB agar + CAF + IPTG 1 mM. PCR of the samples 2 and 5 + control gel.<br>RESULTS: 2 negative, 5 to be confirmed with digestion.",

"description":" ",

"url":"#"

},

{

"date":"1378886400000", /*11 settembre*/     

"type":"demo",

"title":"Miniprep of the constructs 2, 3, 4, 5, 13.<br>Control gel with the previous minipreps and the digested constructs 1 and 6 (loading order: M; 1A; 1D; 1F; 1G; 1I; N; 6B; 6D; 6E; 6F; 6G; 6M; 13 mini; 2 mini; 3 mini; 4 mini; 5mini).<br>RESULTS: the samples 1F and 1N are positive; minipreps quantification on gel: 13: 100 ng/ul; 2: 100 ng/ul; 3: 100 ng/ul; 4: 200 ng/ul; 5: 100 ng/ul.<br>PCR of the samples 1N and 1F.<br>RESULTS: confirmed the positivity of the digestions.<br>Control gel.<br>RESULTS: 1F best sample.",

"description":" ",

"url":"#"

},

{

"date":"1378800000000", /*10 settembre*/     

"type":"demo",

"title":"Boiling prep of the constructs 1 and 16.<br>Control gel (loading order: M; 1A; 1B; 1C; 1D; 1E; 1F; 1G; 1H; 1I; 1L; 1M; 1N; 6A; 6B; 6C; 6D; 6E; 6F; 6G; 6H; 6I; 6L; 6M; 6N).<br>RESULTS: samples chosen for the the double digestion with Eco and Pst: 1A, 1D, 1F, 1G, 1I, 1N, 6B, 6D, 6E, 6F, 6G, 6M.<br>Digestion.<br>Digested samples stored at -20°C.",

"description":" ",

"url":"#"

},

{

"date":"1378713600000", /*09 settembre*/     

"type":"demo",

"title":"PCR of the following samples: 6A, 6C, 6E, 6F, 6G, 6H, 6I, 6L, 6M, 6N, 6P, 6Q.<br>Double digestion (with Eco and Pst) of the 1D and 13.3 samples.<br>Control gel with mini, PCR and digestion samples (loading order: M; 7 mini; 8 mini; 9 mini; 1D digested; 13.3 digested; and the previous PCR samples in the same order).<br>RESULTS: 7 mini, 8 mini, 9 mini: 50 ng/ul; the construct 1 is absent; the sample 13.3 is good; no PCR of the construct n.6 is positive.<br>Inoculum of the 13.3 sample (KAN) for miniprep.<br>Inoculum of the constructs 1 and 16 from plates for boiling prep.",

"description":" ",

"url":"#"

},

{

"date":"1378454400000", /*06 settembre*/     

"type":"demo",

"title":"PCR of the construct n.1 from 4/09 boiling preps: 1A, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1L, 1O, 1Q, 1P.<br>Control gel (following the previous order).<br>RESULTS: the sample 1D is positive.",

"description":" ",

"url":"#"

},

{

"date":"1378368000000", /*05 settembre*/     

"type":"demo",

"title":"PCR of the boling samples 1B, 1M, 1N, 1 1⁄2 A, 1 1⁄2 B, 1 1⁄2 Q, 6B, 6D, 6O, 7a, 7c, 8a (primers: n.408-409).<br>Control gel (following the previous order).<br>RESULTS: no positive results.<br>Inocula of the 7c, 8a and 9b samples.<br>Double digestion (with Eco and Pst) of the 1B, 1M, 1N, 1 1⁄2 A, 1 1⁄2 B, 1 1⁄2 Q, 6B, 6D, 6O samples + control gel (loading order: M; 1B; 1M; 1N; 6B; 6D; 6O; 1 1⁄2 A; 1 1⁄2 B; 1 1⁄2 Q).<br>RESULTS: confirmed the negative results of the PCRs.",

"description":" ",

"url":"#"

},

{

"date":"1378281600000", /*04 settembre*/     

"type":"demo",

"title":"Boiling prep of the following constructs: 1, 1 1⁄2, 6, 7, 8, 9.<br>Control gel (loading order: M; 1A; 1B; 1C; 1D; 1E; 1F; 1G; 1H; 1I; 1L; 1M; 1N; 1O; 1Q; 1P; 1 1⁄2 A; 1 1⁄2 B; 1 1⁄2 C; 1 1⁄2 D; 1 1⁄2 E; 1 1⁄2 F; 1 1⁄2 G; 1 1⁄2 H; 1 1⁄2 I; M; 1 1⁄2 L; 1 1⁄2 M; 1 1⁄2 N; 1 1⁄2 O; 1 1⁄2 Q; 6A; 6B; 6C; 6D; 6E; 6F; 6G; 6H; 6I; 6L; 6M; 6N; 6O; 6P; 6Q; 7a; 7c; 8a; 9b).<br>RESULTS: quantification on gel: 1: 50 ng/ul; 1 1⁄2: 100 ng/ul; 6: 200 ng/ul; 7a, 7c: 50 ng/ul; 8a: 50 ng/ul.",

"description":" ",

"url":"#"

},

{

"date":"1378195200000", /*03 settembre*/     

"type":"demo",

"title":"Control PCRs of 5 mini, 12a 200 ng/ul, 12c 300 ng/ul, 7b, 8a, 8b, 9a, 9b, 9c, 14 plate C, 14 plate D, 16A, 16B, 16C, 16E, 16F, 16G, 16H, 16I, 16L, 16M, 16N, 16O, 16P, 16Q, 16 R.<br>Control gel (following the previous order).<br>RESULTS: positive samples: 5 mini, 9b, 9c; the other samples are all negative.<br>Inocula of 15 colonies per plate (1, 1 1⁄2, 6) + inoculum of the 9b sample for miniprep to be sequenced.<br>Inocula of the 7a, 7b, 8a samples.",

"description":" ",

"url":"#"

},

{

"date":"1378108800000", /*02 settembre*/     

"type":"demo",

"title":"Boiling prep: J04500 (1,2,3,4,5); 7b; 8 (a,b,c); 9 (a,b,c); 14 plate (A,B,C,D,E); 14 reinoculum (A,B,C); 15 (B,D,E); 15a (A,D); 16 (A÷R).<br>Control gel (loading order: M; J04500 (1); J04500 (2); J04500 (3); J04500 (4); J04500 (5); 7b; 8a-b-c; 9a-b-c; 14 plate A-B-C-D-E; 14 reinoculum A-B-C; 15 B-D-E; 15a A-D; 16 A÷R –except the sample D-).<br>RESULTS: good boiling profile.<br>Boiling prep quantification: 7b: 80 ng/ul; 8a: 80 ng/ul; 8b: 80 ng/ul; 9a: 400 ng/ul; 9b: 50 ng/ul; 9c: 400 ng/ul; 14 plate C: 400 ng/ul; 14 plate D: 150 ng/ul; 16A: 20 ng/ul; 16B: 80 ng/ul; 16C: 100 ng/ul; 16E: 20 ng/ul; 16F: 100 ng/ul; 16G: 50 ng/ul; 16H: 60 ng/ul; 16I: 80 ng/ul; 16L: 50 ng/ul; 16M: 50 ng/ul; 16N: 50 ng/ul; 16O: 100 ng/ul; 16P: 100 ng/ul; 16Q: 100 ng/ul; 16R: 100 ng/ul.<br>Ligation of the constructs 1, 1 1⁄2 (J04500 + nikR), 6.<br>Transformation and plate on LB agar + related antibiotics.<br>Inocula of BL21 and DH5α cells from preinocula.",

"description":" ",

"url":"#"

},

{

"date":"1378022400000", /*01 settembre*/     

"type":"demo",

"title":"Inoculum construct n. 16 in LB broth.",

"description":" ",

"url":"#"

},

{

"date":"1377849600000", /*30 agosto*/     

"type":"demo",

"title":"RESULTS 1: yesterday’s plates are empty (except the plate n.16?).<br>PCR of nikR-TER N3, nikR-TER V1, nikR-TER V4 (primers: 408-409), nikR-TER N3, nikR-TER V1, nikR-TER V4 (primers: 395-396).<br>RESULTS 2: positive amplification.",

"description":" ",

"url":"#"

},

{

"date":"1377763200000", /*29 agosto*/     

"type":"demo",

"title":"Ligase inactivation at 80°C for 20 minutes, then immediately in ice.<br>Transformation and plate on LB agar + AMP.<br>Resuspension of the primers: #408: VF2 in 253 ul; #409: VR in 282 ul.<br>Various PCR reactions, GEL RESULTS: 2 midi (985 bp): 100 ng/ul, OK; 3 midi (1076 bp): 100 ng/ul, OK; 4 midi (996 bp): 500 ng/ul, OK; 5 midi (1196 bp): 100 ng/ul, NO; 5 boiling (1196 bp): 500 ng/ul, OK; 6’ (1018 bp): 500 ng/ul, NO; 6’’ (1018 bp): 400 ng/ul, NO; 6 (1018 bp): 600 ng/ul, NO; 7 (2061 bp): 500 ng/ul, OK; 8 (1981 bp): 500 ng/ul, OK; 9 (2181 bp): 500 ng/ul, OK; 12 (1156 bp): 500 ng/ul, NO; 13.3 (716 bp): 400 ng/ul, OK; 14 (374 bp): 300 ng/ul, OK; 15b (386 bp): 300 ng/ul, NO; 15d (386 bp): 100 ng/ul, NO; 15e (386 bp): 300 ng/ul, NO; 15a A (594 bp): 100 ng/ul, OK; 15a D (594 bp): 400 ng/ul, OK; 6.1 boiling (1018 bp): 700 ng/ul, NO; 6.2 boiling (1018 bp): 700 ng/ul, NO; 6.3 boiling (1018 bp): 700 ng/ul, NO; 6.4 boiling (1018 bp): 700ng/ul, NO.",

"description":" ",

"url":"#"

},

{

"date":"1377676800000", /*28 agosto*/     

"type":"demo",

"title":"Boiling prep of the constructs 1, 6, 16 and digestion.<br>Control gel for prep and digestions samples (loading order: 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 6.1; 6.2; 6.3; 6.4; 16 (preps); M; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 6.1; 6.2; 6.3; 6.4; 16 (digestions)).<br>RESULTS: no positive samples (empty plasmids).<br>Rebuilt the constructs 1, 6, 16 starting from the individual parts (O.N. ligation with INVITROGEN T4 DNA LIGASE + 5X BUFFER; final volume: 10 ul; 2 ul per part + plasmid [10 ng/ul] + 2 ul buffer + 1 ul ligase).",

"description":" ",

"url":"#"

},

{

"date":"1377590400000", /*27 agosto*/     

"type":"demo",

"title":"Inocula of the yesterday’s plates (constructs 1, 6, 16).",

"description":" ",

"url":"#"

},

{

"date":"1377504000000", /*26 agosto*/     

"type":"demo",

"title":"Preparation of LB + AMP plates.<br>Cells precipitation and boiling (construct n.6) from the 08/08 inocula (4 samples).<br>Control gel of all the constructs (loading order: M; 2 midi; 3 midi; 4 midi; 5 midi; 5 boiling; 6’; 6’’; 6 (pLac-RBS-LuxI-TER); 7; 8; 9; 12b; 13.3; 14; 15b; 15d; 15e; 15a A; 15a D; M; 6.1; 6.2; 6.3; 6.4).<br>RESULTS: quantification on gel (in ng/ul): 2: [100]; 3: [100]; 4: [500]; 5: [100]; 5 boiling: [500]; 6’: [500]; 6’’: [400]; 6: [500]; 7: [500]; 8: [500]; 9: [500]; 12b: [500]; 13.3: [400]; 14: [300]; 15b: [300]; 15d: [400]; 15e: [300]; 15a A: [100]; 15a D: [100]; 6 (1;2;3;4): [700].<br>Ligation of the constructs n. 1, 6, 16 with PSB1A3 plasmid (already linearized), transformation of DH5α cells and plate on LB agar + AMP.",

"description":" ",

"url":"#"

},

{

"date":"1376035200000", /*09 agosto*/     

"type":"demo",

"title":"Inocula miniprep (constructs n.4,6) and boiling prep (costruct n.16).<br>Digestion with EcoRI and PstI.<br>Control gel (loading order: 1-11: construct n.16; 12-13: constructs n.4-6; the same order for the digestions).<br>RESULTS: the constructs n.16 is absent, the n.4 is present and the n.6 has a negative digestion profile (redo).<br>Ten J04500 colonies replated on LB agar + CAF.",

"description":" ",

"url":"#"

},

{

"date":"1375948800000", /*08 agosto*/     

"type":"demo",

"title":"Prep of construct n.16 + control gel.<br>RESULTS: fragments at 2000 and 300 bp. Inocula of construct n.4 (clone 4-3), 6 (clone 6-5) and 16 in LB broth + AMP.<br>Transformation of DH5α cells with the construct n.1 (1 assembled with NEB ligase, 1 with Invitrogen ligase) and plate on LB agar + IPTG + AMP.",

"description":" ",

"url":"#"

},

{

"date":"1375862400000", /*07 agosto*/     

"type":"demo",

"title":"Boiling prep of the construct n.10.<br>RESULTS: failed. RESULTS of the transformation reactions: constructs n.10 and 16 are negative, only an inoculum of the construct n.16 seems to be positive.<br>Ligation and cloning of the construct n.1 starting from resuspended J04500, because the control gel has shown that the boiling prep of J04500 was degraded.<br>Transformation.<br>The constructs n.4 (clone 4-3) and 6 (clone 6-5) have been plated on related LB agar + IPTG, in order to avoid pink colonies’ contamination.<br>Rebuilt the construct 13 +16 (n.17) (digestion, ligation, cloning and transformation).",

"description":" ",

"url":"#"

},

{

"date":"1375776000000", /*06 agosto*/     

"type":"demo",

"title":"Inocula boiling prep: construct n.8, 15, 15A, 11A, 11C + control gel.<br>Single digestion (with EcoRI) of the construct n.8, double digestion for the other samples + control gel.<br>RESULTS: samples chosen as positive: 8A, 15A a, 15B, 15A, 15C. Assembly of the constructs.<br>Transformation of DH5α cells with 6+1 (construct n.10) in PSB1C3, 13+15 (construct n.16) in PSB1A3 and plate on LB agar + IPTG + related antibiotics.<br>Inoculum of 6+1 (construct n.10) from an old plate to verify that the old ligation was good, otherwise we can use the new plates (today).",

"description":" ",

"url":"#"

},

{

"date":"1375689600000", /*05 agosto*/     

"type":"demo",

"title":"Boiling prep + digestion with EcoRI and PstI + control gel (single digestion for the samples 17A, B, C: 17A E, 17A P, 17B E, 17B P, 17C E, 17C P) (double digestion for the other samples). (loading order: M; 11A-1; 11A-2; 11A-3; 11C-1; 11C-2; 11C-3; 13-1; 13-2; 13-3; 15-1; 15-2; 15-3; 15A-1; 15A-2; 15A-3; 17-1P; 17-2P; 17-3P; 17-1E; 17-2E; 17-3E; CTRL AMP; CTRL KAN).<br>RESULTS: The constructs n.13 and 17 only have a positive digestion profile. Inocula of 11A, 11C, 15, 15A, 4+2 (construct n.8) in LB broth + related antibiotics (AMP, KAN, CAF) (6 tubes per construct).",

"description":" ",

"url":"#"

},

{

"date":"1375603200000", /*04 agosto*/     

"type":"demo",

"title":"Inocula aliquoted in eppendorfs and stored at 4°C.<br>The remaining broth was centrifuged, obtaining a pellet stored at -20°C.",

"description":" ",

"url":"#"

},

{

"date":"1375516800000", /*03 agosto*/     

"type":"demo",

"title":"Inocula of the last plates.",

"description":" ",

"url":"#"

},

{

"date":"1375430400000", /*02 agosto*/     

"type":"demo",

"title":"Boiling prep of yesterday’s inocula + control gel (order: M, 3+2, 4+2, 5+2, 6+1, 12, 14, C0261).<br>RESULTS: chosen as positive the third sample of each construct.<br>Digestion with EcoRI and PstI + control gel (loading order: M, 3+2, 4+2, 5+2, 6+1, 12, 14, CTRL PSB1C3 0,8 ug/ul).<br>RESULTS: profile not satisfactory → Digestion of all boiling products with EcoRI and PstI + control gel (same loading order).<br>RESULTS: chosen 3+2: C; 4+2: none; 5+2: A; 6+1: B; 12: B; 14: A.<br>Preparation of 10 LB + AMP tubes (9 + CTRL; 5 ml/tube) and 13 LB + KAN tubes (12+ CTRL; always 5 ml/tube).<br>Ligations: 10 E/S + 7 X/P + PSB1A3 E/P → 11A; 10 E/S + 9 X/P + PSB1A3 E/P → 11C; 1 E/S + 12 X/P + PSB1K3 E/P → 17; B0034 E/S + 14 X/P + PSB1K3 E/P → 15; R0062 E/S + C0261 X/P + PSB1K3 E/P → 13; J04500 E/S + 14 X/P + PSB1K3 E/P → 15A.<br>Transformation of DH5α cells with the various ligated plasmids and plate on LB + related antibiotics.",

"description":" ",

"url":"#"

},

{

"date":"1375344000000", /*01 agosto*/     

"type":"demo",

"title":"Miniprep of the constructs to be sequenced (1,2,3,4,5,6) + control gel.<br>RESULTS: 1: 100 ng/ul; 2: 80; 3: 100; 4: 100; 5: 80; 6: 100.<br>Yesterday’s ligations + C0261 inocula (3 tubes per construct).",

"description":" ",

"url":"#"

},

{

"date":"1375257600000", /*31 luglio*/     

"type":"demo",

"title":"Ligations: 5 + 2 in PSB1C3, 3 + 2 in PSB1C3, 4 + 2 in PSB1C3, 6 + 1 in PSB1C3, construct n.12 (pUreA + I0462 in PSB1C3), construct n.14 (Hpn + B0015 in PSB1C3).<br>Transformation of DH5α cells with previous ligated plasmids and with C0261; plate on LB agar + CAF and + AMP (for C0261).<br>Inocula for miniprep to be sequenced: construct n.1 (boiling n.13), construct n.2 (boiling n.25), construct n.3 (boiling n.35), construct n.4 (boiling n.43), construct n.5 (boiling n.52) and construct n.6 (boiling n.65).",

"description":" ",

"url":"#"

},

{

"date":"1375171200000", /*30 luglio*/     

"type":"demo",

"title":"Boiling prep of yesterday’s inocula + control gel.<br>Boiling digestion with EcoRI and PstI + control gel.<br>RESULTS: chosen as positive the following samples: boiling n.13 ([ ] = 200 ng/ul), boiling n.25 ([ ] = 100 ng/ul), boiling n.35 ([ ] = 150 ng/ul), boiling n.43 ([ ] = 150 ng/ul), boiling n.52 ([ ] = 100 ng/ul), boiling n.65 ([ ] = 200 ng/ul).",

"description":" ",

"url":"#"

},

{

"date":"1375084800000", /*29 luglio*/     

"type":"demo",

"title":"Preparation of LB broth + related antibiotics for inocula.<br>Ligation inocula (pLac-RBS-nikR-TER in PSB1A3, pUreA-K773003 in PSB1K3, pexbB-E0240 in PSB1A3, pnik-E0240 in PSB1A3, pint-E0240 in PSB1A3, pUreA-K805016 in PSB1A3).<br>Inocula of I0462 e R0062 in LB + AMP (3 +2 LB + AMP tubes; final volume: 5 ml per tube).",

"description":" ",

"url":"#"

},

{

"date":"1374825600000", /*26 luglio*/     

"type":"demo",

"title":"RESULTS inocula for boiling: inocula failed.<br>RESULTS plate (pUreA + K805016 + PSB1A3): majority of red colonies, some white. pLAC-RBS + nikR-TER + PSB1A3, pUreA + K773003 + PSB1K3, pexbB + E0240 + PSB1A3, pnik + E0240 + PSB1A3, pint + E0240 + PSB1A3: ligation (parts already digested).<br>Preparation of LB + AMP and KAN plates.<br>Transformation of DH5α cells with ligated plasmids and plate on LB agar + related antibiotics.<br>PCR of all promoters + control gel (1,2: intergenic region; 3,4: pnikR; 5,6: pexbB).<br>RESULTS: all the amplifications have been successful!",

"description":" ",

"url":"#"

},

{

"date":"1374739200000", /*25 luglio*/     

"type":"demo",

"title":"Boiling prep of yesterday’s inocula (K805016) + control gel (K80 1, K80 2, K80 R1, K80 R2).<br>RESULTS: we choose the re-inocula for ligation.<br>Preparation of LB + CAF plates. Digestion of K805016 with XbaI and PstI.<br>Ligation pUreA (already digested) + K805016 + PSB1A3 (already digested).<br>Transformation of DH5α cells with ligated plasmid and plate on LB agar + AMP + IPTG.<br>Inocula plates (5 inocula per 5 plates= 25 tubes).",

"description":" ",

"url":"#"

},

{

"date":"1374652800000", /*24 luglio*/     

"type":"demo",

"title":"Inocula boiling prep.<br>New and old digestions NikR + TER (N3, N4, V1, V4) + control gel.<br>pLAC-RBS + nikR-TER + PSB1A3, pUreA + K773003 + PSB1K3, pexbB + E0240 + PSB1A3, pnik + E0240 + PSB1A3, pint + E0240 + PSB1A3: digestion and ligation.<br>Inocula of K805016 from plate in 2 tubes (3 ml per tube) and other 2 (3 ml/tube, always K805016) from previous inoculum. Transformation of DH5α cells with all ligation products.<br>Plate of Hpn and pUreA re-inocula (LB + CAF + 20 ul cells + 100 ul IPTG 1mM).",

"description":" ",

"url":"#"

},

{

"date":"1374566400000", /*23 luglio*/     

"type":"demo",

"title":"Boiling of yesterday’s inocula + control gel.<br>RESULTS: (photo).<br>Control gel for NikR PCR reaction ([amplified NikR DNA sequence] =100 ng/ul).<br>Ligation plates inocula.",

"description":" ",

"url":"#"

},

{

"date":"1374480000000", /*22 luglio*/     

"type":"demo",

"title":"Digestion and ligation of NikR + TER in PSB1C3 (new digestions). Ligation NikR + TER (old digestions) in PSB1C3 (new digestions).<br>Digestions called NEW and OLD plated on LB agar with CAF 35 ug/ml + IPTG 1mM. Inoculum for boiling of E0240, J04500, K773003, K805016, Hpn and pUreA (5 colonies per part).<br>New inoculum of 3 inocula for boiling (11/07/13).<br>PCR of NikR for new purification, positive result. CryoTEM analysis: failed.",

"description":" ",

"url":"#"

},

{

"date":"1374220800000", /*19 luglio*/

"type":"demo",

"title":"Boiling prep of Hpn and pUreA inocula.<br>RESULTS: no plasmids.",

"description":" ",

"url":"#"

},

{

"date":"1374134400000", /*18 luglio*/

"type":"demo",

"title":"RESULTS: empty gel.<br>Resuspension of Hpn and pUreA (in 400 ul TE) (+ control gel: ladder, PSB1C3 0,8 ug/ul, Hpn, pUreA).<br>RESULTS: no Hpn and pUreA on gel.<br>New gel with Hpn and pUreA.<br>RESULTS: no results.<br>Photometric quantification of plasmid DNA (Hpn: 1086,4 ug/ml; pUreA: 1288,9 ug/ml; 260 nm: Hpn 546; pUreA 900; 260/280: Hpn 2,06; pUreA 2,23).<br>RESULTS: yesterday’s inocula contained only red colonies (boiling prep failed).<br>Gel to control the maxiprep aliquot of PSB1C3 (ladder, PSB1C3 aliquot of use, PSB1C3 from maxiprep, PSB1C3 0,7 ug/ul) 0,8 ug/ul.<br>10 inocula of Hpn and 10 of pUreA in LB + CAF (5 ml per tube).",

"description":" ",

"url":"#"

},

{

"date":"1374048000000", /*17 luglio*/

"type":"demo",

"title":"Cryo-TEM assay (make another one 22/7).<br>Maxiprep of Hpn and pUreA inocula (DNA precipitation O.N. at -20°C).<br>Gel to value plasmids integrity (Ladder, Control, E0240, J04500, K773003, K805016).<br>Ligation E0240 + pnik, pexbB, pint. Inoculum NikR + TER colonies.",

"description":" ",

"url":"#"

},

{

"date":"1373961600000", /*16 luglio*/

"type":"demo",

"title":"RESULT: No colonies.<br>Ligation NikR + TER, transformation and plate on LB agar + CAF + IPTG.<br>Maxiprep of yesterday’s inocula (DNA precipitation 0.N. at -20°C).<br>Inocula of pUreA and Hpn (2 X 100 ml LB broth; 2 X 280 ul CAF).",

"description":" ",

"url":"#"

},

{

"date":"1373875200000", /*15 luglio*/

"type":"demo",

"title":"Control gel of last maxipreps (E0240 W2, K773003, K805016, J04500, control: PSB1C3 0,7ug/ul). RESULTS: empty gel.<br>Transformation DH5α cells with ligated plasmid containing NikR + TER, and plate on LB agar + CAF + IPTG (only one plate).<br>Inoculum of E0240, K805016, K773003, J04500 in LB broth + CAF.<br>ATR assay with samples incubated with PBS buffer.<br>RESULTS: low proteic signal (probably a large amount of protein was lost because of dialysis, cut-off value?).<br>Make another ATR assay using Tris (the Phosphate Buffer precipitates nickel!).",

"description":" ",

"url":"#"

},

{

"date":"1373616000000", /*12 luglio*/

"type":"demo",

"title":"Ligation NikR + TER (in CAF).<br>Preparation of LB plates for future clonings.<br>Revision of cloning and ligation schemes.",

"description":" ",

"url":"#"

},

{

"date":"1373529600000", /*11 luglio*/

"type":"demo",

"title":"Gel electrophoresis of yesterday’s digestion.<br>Inocula maxi prep.<br>Photometric quantification of plasmid DNA.<br>Gel electrophoresis of Hpn and pUreA boiling samples.<br>Sephadex column and NikR samples stored at -20°C.",

"description":" ",

"url":"#"

},

{

"date":"1373443200000", /*10 luglio*/

"type":"demo",

"title":"Boiling prep of Hpn and pUreA inocula. Control gel.<br>Digestion with EcoRI and PstI + control gel.<br>Inocula of yesterday’s colonies in LB broth + IPTG + related antibiotics.<br>Samples preparation for ATR and Cryo-TEM.<br>Recovery of 120 ul from dialysis device.<br>2 X 20 ul aliquots of untreated sample + 2 X 10 ul aliquots treated with nickel (final volume: 50 ul: 10 ul NikR 1 ug/ul + 3 ul nickel sulfate + 37 ul PBS with 10mM phosphate).",

"description":" ",

"url":"#"

},

{

"date":"1373356800000", /*9 luglio*/

"type":"demo",

"title":"ICP-AES assay (RESULTS: direct proportionality between [NikR] and [Ni]), NikR quantification by Biorad method.<br>Preparation of LB plates (2X500ml AMP, 1X200ml CAF, 1X200ml KAN).<br>Incubation drafting with EDTA + DTT and dialysis against PBS O.N. Red-white screen on UV lamp.<br>Inocula of yesterday’s colonies in LB broth + CAF (6 samples for HPN and 6 for pUreA).<br>Transformation of DH5α cells with plasmids containing pLAC-RBS (J04500), RBS-RFP-LVA-TER (K773003), RBS-GFP-TER (E0240), RBS-LuxI-TER (K805016), RBS-LuxR-TER (I0462), LuxPr (R0062) and plate on LB + IPTG + related antibiotics.",

"description":" ",

"url":"#"

},

{

"date":"1373270400000", /*8 luglio*/

"type":"demo",

"title":"future clonings organization.<br>Resuspension GBlocks.<br>Digestion of PSB1C3 and GBlocks with EcoRI and PstI.<br>Ligation.<br>Transformation of DH5α cells with ligated plasmids and plate on LB agar + CAF + IPTG (2 plates).<br>NikR purification by Ni-NTA resin for new assay, new protocol (new ATR assay).",

"description":" ",

"url":"#"

},

{

"date":"1373011200000", /*5 luglio*/

"type":"demo",

"title":"ATR measurement assay.<br>Inocula boiling prep + control gel.",

"description":" ",

"url":"#"

},

{

"date":"1372924800000", /*4 luglio*/

"type":"demo",

"title":"boiling prep digestion control + gel.RESULTS: 210 bp fragment obtained.<br>Boiling and digestion gel electrophoresis.<br>Packing columns and sample chromatography (photos).<br>Samples delivered in chemistry lab for ICP-AES essay.<br>Team meeting.<br>Screening white colonies on UV lamp.<br>Inocula of white colonies.",

"description":" ",

"url":"#"

},

{

"date":"1372838400000", /*3 luglio*/

"type":"demo",

"title":"Boiling prep and control agarose gel electrophoresis.<br>Study of the digestion profile.<br>Resin preparation and new ICP-AES protocol drafting.<br>NikR incubation with nickel sulfate on lab wheel at 4°C O.N (for ICP and ATR).",

"description":" ",

"url":"#"

},

{

"date":"1372752000000", /*2 luglio*/

"type":"demo",

"title":"Red-White screen.<br>RESULTS: white colonies obtained.<br>Preparation of LB broth and inoculum into tubes.<br>Protein quantification by Biorad method.",

"description":" ",

"url":"#"

},

{

"date":"1372665600000", /*1 luglio*/

"type":"demo",

"title":"IPTG (1mM) added to KAN plates.<br>Digestion and ligation.<br>Transformation and plate on KAN +IPTG medium.<br>NikR purification by Ni-NTA resin.",

"description":" ",

"url":"#"

},

{

"date":"1372406400000", /*28 giugno*/

"type":"demo",

"title":"Control AGE (B0015, R0010, B0034, PSB1K3, PSB1C3 and PSB1A3).<br>Digestion and ligation protocol drafting.<br>Nickel binding essay protocol modified.",

"description":" ",

"url":"#"

},

{

"date":"1372320000000", /*27 giugno*/

"type":"demo",

"title":"Maxiprep of B0015, R0010, B0034, PSB1K3.<br>Photometric quantification of plasmid DNA.<br>GBlocks (HPN, pUreA) now available.",

"description":" ",

"url":"#"

},

{

"date":"1372233600000", /*26 giugno*/

"type":"demo",

"title":"Maxiprep.<br>Preparation of LB broth.<br>Inoculum.<br>Team meeting.",

"description":" ",

"url":"#"

},

{

"date":"1372147200000", /*25 giugno*/

"type":"demo",

"title":"Inoculum of PSB1C3 e PSB1A3.",

"description":" ",

"url":"#"

},

{

"date":"1372060800000", /*24 giugno*/

"type":"demo",

"title":"Lab team meeting.",

"description":" ",

"url":"#"

},

{

"date":"1371801600000", /*21 giugno*/

"type":"demo",

"title":"Preparation of the related pellets (B0015 in Amp, B0034 in Amp, R0010 in CF), then stored at -20°C.",

"description":" ",

"url":"#"

},{

"date":"1371456000000", /*17 giugno*/

"type":"demo",

"title":"Inoculum in LB broth.",

"description":" ",

"url":"#"

},

{

"date":"1371196800000", /*14 giugno*/

"type":"demo",

"title":"preparation of LB plates.<br>Transformation of DH5α cells with plasmids containing pLac, RBS and the terminator + various controls.<br>Plate cells.",

"description":" ",

"url":"#"

},

{

"date":"1371110400000", /*13 giugno*/

"type":"demo",

"title":"Maxiprep.",

"description":" ",

"url":"#"

},

{

"date":"1371024000000", /*12 giugno*/

"type":"demo",

"title":"RESULTS: red colonies obtained (photo).<br>Inoculum in LB broth.<br>GBlocks sequence control.",

"description":" ",

"url":"#"

},

{

"date":"1370937600000", /*11 giugno*/

"type":"demo",

"title":"RESULTS: few colonies on CAF plates.<br>Streaking colonies on similar plates with related antibiotics and IPTG as inducer. O.N. incubation.",

"description":" ",

"url":"#"

},

{

"date":"1370851200000", /*10 giugno*/

"type":"demo",

"title":"Transformation of DH5α cells with four plasmids containing RFP, with different resistance, and plate on four different LB culture media with the related antibiotics (A-K-T-C). O.N incubation.",

"description":" ",

"url":"#"

},

{

"date":"1370592000000", /*7 giugno*/

"type":"demo",

"title":"RESULTS: no growth.<br>These plasmids must be processed before proceeding with the transformation.",

"description":" ",

"url":"#"

},

{

"date":"1370505600000", /*6 giugno*/

"type":"demo",

"title":"Choice colonies and inoculum in LB broth.",

"description":" ",

"url":"#"

},

{

"date":"1370419200000", /*5 giugno*/

"type":"demo",

"title":"Preparation of A-K-T-C plates and transformation of DH5α competent cells with four plasmids of PSB1 serie.<br>Plate and O.N. incubation.<br>PCR of pexbB and NikR (new DNA aliquots).<br>RESULTS: pexbB amplified correctly (few products), nikR amplified but with a nonspecific lower band.",

"description":" ",

"url":"#"

},

{

"date":"1370332800000", /*4 giugno*/

"type":"demo",

"title":"PCR of four samples (two for pexbB, two for NikR).<br>Control gel.<br>RESULTS: formation of nonspecific products (consider reasons; Taq?).",

"description":" ",

"url":"#"

},

{

"date":"1370246400000", /*3 giugno*/

"type":"demo",

"title":"PCR of two pexbB samples and NikR.<br>AGE and eluition.<br>RESULTS: few amplification products.",

"description":" ",

"url":"#"

}

];

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