Line 4:
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===Visualization===
+ ====Ligation of Gblock 2-2====
+ ''By Caroline and Lea''
+
+ TODOà remplir
+
+ ====Transformation of Gblocks in DH5α====
+ ''By Caroline and Lea''
+
+ 6 transformations were performed with the ligation products containing plasmids: pPS16_001, pPS16_002, pPS16_003, pPS16_004, pPS16_005, pPS16_006. 3 other transformations were performed with plasmids pPS16_007, pPS16_008 and pPS16_009 extracted the 30/06/16. TODOajouter le lien The transformation protocol were followed increasing plasmids quantities (5µL instead of 1µL).
+ After transformation, cells are spread on petri dishes containing LB + Ampicillin (50µg/mL) + Xgal + IPTG.
+
====Digestion of plasmids====
====Digestion of plasmids====
''By Mathilde and Alice''
''By Mathilde and Alice''
Revision as of 15:24, 7 July 2016
Monday 4th July Lab work Visualization Ligation of Gblock 2-2 By Caroline and Lea
TODOà remplir
Transformation of Gblocks in DH5α By Caroline and Lea
6 transformations were performed with the ligation products containing plasmids: pPS16_001, pPS16_002, pPS16_003, pPS16_004, pPS16_005, pPS16_006. 3 other transformations were performed with plasmids pPS16_007, pPS16_008 and pPS16_009 extracted the 30/06/16. TODOajouter le lien The transformation protocol were followed increasing plasmids quantities (5µL instead of 1µL).
After transformation, cells are spread on petri dishes containing LB + Ampicillin (50µg/mL) + Xgal + IPTG.
Digestion of plasmids By Mathilde and Alice
The following plasmids were digested again with EcoR1 and HindIII using this protocol :
pPS16_001 (from the 6 clones that were selected on 29/06/16)
pPS16_003 (from the 6 clones that were selected on 29/06/16)
pPS16_005 (from the 6 clones that were selected on 29/06/16)
pPS16_006 (from the 6 clones that were selected on 29/06/16)
Component
Volume (µL)
Plasmid
10
Red buffer 10x
2
Water
7
EcoRI enzyme
0.5
HindIII enzyme
0.5
The digestion products were mixed as follow to be migrated on an agarose gel.
Component
Volume (µL)
Digested DNA
20
Loading buffer
3.3
The DNA concentration was still insufficient meaning the extraction step was not efficient.
Bringing DNA closer By Naiane and Laetitia
Plasmids from the following strains were extracted with the kit "Charge Switch-Pro Plasmid Miniprep":
DS-NMcas
DS-SPcasN-
DS-ST1casN-
DS-TDcasN- Then plasmids were digested with AvrII
Component
Volume (µL)
Plasmid
10
Tango buffer 10x
2
Water
7
AvrII enzyme
1
The mix was incubated at 37°C for 1 hour.
An electrophoresis was done (0.8% of agarose).
Component
Volume (µL)
Digested DNA
5
Water
5
Loading buffer
2
