Team:Paris/September 12

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Checking our ligases (following)

Analysis of the ligation products by electrophosesis

1% agarose gel

Ligation of the plasmid digested by EcoRI
Ligation of the plasmid digested by BspHI

Analysis of the ligation of the plasmid digested by EcoRI
No heat inactivation of the ligase

well n° 1 2 3 4 5 6 7 8 9 10 11 12 13
sample 1 kb ladder circular
plasmid (MP101)
circular
plasmid (MP101)
digested
plasmid (D202)
digested
plasmid (D202)
1 2 3 4 5 6 7 100 bp ladder
purification
digestion products
gel extraction column no
purification
ligase used L1 L2 L3 L1 L2 L3 L1
amount loaded 10 µL 0,5 µL 1 µL 2 µL 3 µL 10 µL 3 µL 10 µL

Analysis of the ligation of the plasmid digested by BspHI
Heat inactivation of the ligase (20 min at 65°C)

well n° 1 2 3 4 5 6 7 8 9 10 11
sample 1 kb ladder circular
plasmid (MP101)
circular
plasmid (MP101)
digested
plasmid (D203)
8 9 10 11 12 13 100 bp ladder
purification
digestion products
column no
purification
ligase used L1 L2 L3 L1 L2 L3
amount loaded 10 µL 0,3 µL 0,5 µL 4 µL 15 µL 10 µL 10 µL