Difference between revisions of "Team:Pasteur Paris/Microbiology week10"

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                       <h6><U>Results :</U></h6><br /><br />
 <center><img src = "https://static.igem.org/mediawiki/2016/c/cf/Week_10_152Electrophoresis_with_the_results_of_digestion.jpg"; alt "Gel of the results of digestion"/>
-<i><p> <U>Figure 1:</U> Gel of the results of digestion </i></p></center><br />
+<i><p> <U>Figure 18 :</U> Gel of the results of digestion </i></p></center><br />
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                  </p>
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 <h6><U>Results :</U></h6><br /><br />
 <center><img src = "https://static.igem.org/mediawiki/2016/b/bb/Week_10_154Gel_extraction_of_A1-A2-D1-D2.jpg"; alt "Extraction gel of A1-A2-D1-D2"/>
-<i><p> <U>Figure 3 :</U> Extraction gel of A1-A2-D1-D2 </p></i></center>
+<i><p> <U>Figure 19 :</U> Extraction gel of A1-A2-D1-D2 </p></i></center>
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          </p>
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 <h6><U>Results :</U></h6><br /><br />
 <center><img src = "https://static.igem.org/mediawiki/2016/a/ad/Week_10_161Electrophoresis_of_the_PCR_done_on_the_8th_of_August_with_A1-A2-D1-D2.jpg" ; alt "Electrophoresis gel of the PCR"/>
-<i><p> <U>Figure 2 :</U> Electrophoresis gel of the PCR</p></i></center><br />
+<i><p> <U>Figure 20 :</U> Electrophoresis gel of the PCR</p></i></center><br />
 The PCR works properly since we noticed significant bands at the expected level.<br />
 <br /><br /><br />

Reactants	Volumes (µl)
Xba I	30
Hind III	30
Buffer 2.1	90
Distilled H₂O	90
Total	150

	Mix with two primers	Mix with primer S only	Mix with primer AS only
Takara enzyme (µl)	6	6	6
Primer S (µl)	6	6	Ø
Primer AS (µl)	6	Ø	6
MgCl₂ (µl)	15	15	15
dNTPs (µl)	15	15	15
Buffer (µl)	30	30	30
H₂O (µl)	216	222	222
Total (µl)	294	294	294

	Mix with two primers	Mix with primer S only	Mix with primer AS only
A1	tube 1	tube 2	tube 3
A2	tube 4	tube 5	tube 6
D1	tube 7	tube 8	tube 9
D2	tube 10	tube 11	tube 12

DNA	Colonies	Weight of the gel (mg)
B2	Colony 4	367
Colony 7	432
Colony 9	269
E2	Colony 1	300
Colony 2	355
Colony 3	354
Colony 4	314
Colony 5	299
Colony 6	275
Colony 7	277
E1	Colony 1	404
Colony 2	321

	E1	E2	B2	pET 43.1(a+)
E1 (µl)	15	Ø	Ø	Ø
E2 (µl)	Ø	15	Ø	Ø
B2 (µl)	Ø	Ø	15	Ø
pET 43.1(a+) (µl)	4	4	4	4
Ligase (µl)	1	1	1	1
TOP0 (µl)	2.2	2.2	2.2	2.2
H₂O (µl)	Ø	Ø	Ø	15
V_total (µl)	22.2	22.2	22.2	22.2

	Volumes (µl)
DNA	5
Xba I	1
Hind III	1
Buffer 2.1	2
H₂O	11
Total	20

	Volumes (µl)
Xba I	20
Hind III	20
Buffer 2X	40
H₂O	220
Total	300

	Volumes (µl)
DNA	12.5
Xba I	2
Hind III	4
Buffer CutSmart	5
H₂O	26.5
Total	50

Sample	1	2	3	4	5	6	Time of addition of iPTG	Concentration (ng/µl)
B1 (1)	0.022	0.062	0.152	0.344	0.557	0.694	16 h 55	0.859
B1 (2)	0.185	0.417	0.693				15 h 25	0.956
B1 (3)	0.075	0.211	0.413	0.688			15 h 55	0.920
C2 (1)	0.060	0.166	0.350	0.627	0.699		16 h 25	0.905
C2 (2)	0.044	0.119	0.296	0.510	0.698		16 h 25	0.910
C2 (16)	0.080	0.230	0.445	0.689			15 h 55	0.907

*Table 117*
	B2	E1	E2
NaAc (µl)	15	10	35
Ethanol (µl)	875	250	375

*Table 123*
	Volumes ( µl)
DNA	46
CutSmart	6
H₂O	6.7
rSAP	1.3
Total	60

Difference between revisions of "Team:Pasteur Paris/Microbiology week10"

Latest revision as of 02:20, 20 October 2016

click on the weeks

Microbiology Notebook

Week 10

August 8, 2016:

150. Culture for miniprep of C2 v2 and B1 v2

151. Digestion of B2, E1 and C2 from the 4th of August

152. Electrophoresis with the results of digestion

153. PCR of inserts A1/A2/D1/D2

154. Gel extraction of A1/A2/D1/D2

155. Gel extraction of B2, E1 and E2

156. Resuspension of inserts B2/E1/E2

August 9, 2016:

157. Ligation of inserts B2⁄E1⁄E2 with pET 43.1a(+)

158. Miniprep of C2 v2 (TOP 10) and B1 v2 (TOP 10 )

159. Miniprep preculture of C1 v2 in pET 43.1a(+)

160. Preparation of 10 aliquots of carbenicillin at 50 mg⁄m

161. Electrophoresis of the PCR done on the 8th of August with A1⁄A2⁄D1⁄D2

162. Transformation of A1⁄A2⁄D1⁄D2 in TOP 10

163. Transformation of E1⁄E2 and B2 in TOP 10

August 10, 2016:

164. Miniprep of C1 v2 (culture from the 9 of August)

165. Digestion of C1 v2 before electrophoresis

166. Digestion of pET 43.1 (a+) with Xba I and Hind III

167. Electrophoresis and gel extraction

168. Electrophoresis of C1 digested

169. Transformation of B2 (4, 7 and 9), E1 (1 and 2) and E2 (2, 3, 4, 5, 6 and 7)

170. Aliquot of antibodies 462

171. Culture of A1⁄A2⁄D1⁄D2

172. Ligation of A1⁄A2⁄D1⁄D2 in TOPO

173. Transformation of A1⁄A2⁄D1⁄D2 with TOPO in TOP 10 competent cells

174. Transformation of B1 v2 and C2 v2 in BL21DE3

175. Dosage of digested pET 43.1 (a+)

176. Transformation of C2 v2 and B1 v2 in pET 43.1a(+) and DH3α

August 11, 2016:

177. Absorbance of precultures C2 v2(1, 2, 3) and B1 v2 (1, 2, 16)

178. Dephosphorylation of pET 43.1a(+) digested on the 10th of August

179. Miniprep of B1 v2 ⁄E1⁄E2 in TOPO

180. Transformation of B1 v2 colony 8 and C2 v2 colony 16 in pET 43.1 (a+) and in DH 3α

181. Precultures of B1 v2 and C2 v2

182. Digestion of pET 43.1 (a+) with Xba I and Hind III

August 12, 2016:

183. Miniprep from precultures of B2 v2/E1/E2 in TOPO

184. Digestion of inserts B2 v2/E1/E2 with XbaI and HindIII

185. Culture of C2 v2 and B1 v2 in 1 l of LB

186. Agarose gel to analyze digestion of pET 43.1(a+) done on the 11th of August

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151. Digestion of B2, E1 and C2 from the 4^th of August

161. Electrophoresis of the PCR done on the 8^th of August with A1⁄A2⁄D1⁄D2

178. Dephosphorylation of pET 43.1a(+) digested on the 10^th of August

186. Agarose gel to analyze digestion of pET 43.1(a+) done on the 11^th of August