User:DavidC/18 September 2009
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+ | === Friday the 18th === | ||
+ | |||
+ | ==== Restriction digest ==== | ||
+ | |||
+ | ==== Ligation between B0014 and P1003 ==== | ||
+ | |||
+ | Restriction digest of BBa_B0014 (= 2,41µg/µL) by EcoRI and XbaI (3284bp): <br> | ||
+ | |||
+ | DNA (10µg final) = 4,2µL <br> | ||
+ | Buffer Eco R1 (NEB) = 5µL <br> | ||
+ | H20 = 39,8µL <br> | ||
+ | Eco R1 = 1µL <br> | ||
+ | Incubation 1h at 37°C. <br> | ||
+ | |||
+ | DNA purification with a nucleospin (macherey-nagel). <br> | ||
+ | |||
+ | DNA = 50µL <br> | ||
+ | Buffer 2 (NEB) = 6µL <br> | ||
+ | BSA (NEB) = 0,6µL <br> | ||
+ | H20 = 2,4µL <br> | ||
+ | Xba 1 = 1µL <br> | ||
+ | Incubation 1h at 37°C. <br><br> | ||
+ | |||
+ | Restriction digest of P1003 (4,17µg/µL) by EcoRI and SpeI (967bp): <br> | ||
+ | |||
+ | DNA (10µg final) = 2,4µL <br> | ||
+ | Buffer Eco R1 (NEB) = 5µL <br> | ||
+ | H20 = 40,6µL <br> | ||
+ | BSA = 0,5µL <br> | ||
+ | Eco R1 (NEB) = 1µL <br> | ||
+ | Spe 1 (NEB) = 1µL <br> | ||
+ | Incubation 1h at 37°C. <br> | ||
+ | |||
+ | ==== Ligation of B0014 with C0012 ==== | ||
+ | |||
+ | B0014 (= 2,41µg/µL) restriction digest by EcoRI and XbaI (3284bp): <br> | ||
+ | |||
+ | Same samples as the restriction digest used for B0014 and P1003 ligation. <br><br> | ||
+ | |||
+ | C0012 (1,56µg/µL) restriction digest by EcoRI and SpeI (1128bp): <br> | ||
+ | |||
+ | DNA (10µg final) = 6,4µL <br> | ||
+ | Buffer Eco R1 (NEB) = 5µL <br> | ||
+ | H20 = 36,1µL <br> | ||
+ | BSA = 0,5µL <br> | ||
+ | Eco R1 (NEB) = 1µL <br> | ||
+ | Spe 1 (NEB) = 1µL <br> | ||
+ | Incubation 1h at 37°C. <br> | ||
+ | |||
+ | ==== DNA electrophoresis ==== | ||
+ | |||
+ | 85 Volt, 15 minutes. <br> | ||
+ | 105 Volt, 40 minutes. <br> | ||
+ | Ladder fermentas 1 Kb. <br> | ||
+ | |||
+ | |||
+ | Samples: | ||
+ | |||
+ | ==== DNA purification ==== | ||
+ | |||
+ | Kit Qiagen “gel extraction kit”, final volume = 50µL. <br> | ||
+ | |||
+ | ==== Ligation ==== | ||
+ | |||
+ | Ligation schemes: plasmid / insert: <br> | ||
+ | |||
+ | BBa_B0014/BBa_P1003 ; <br> | ||
+ | BBa_B0014/BBa_C0012. <br> | ||
+ | |||
+ | First report: <br> | ||
+ | Plasmid = 1µL <br> | ||
+ | Insert = 5µL <br> | ||
+ | |||
+ | Second report: <br> | ||
+ | Plasmid = 1µL <br> | ||
+ | Insert = 3µL <br> | ||
+ | |||
+ | Third report: <br> | ||
+ | Plasmid = 1µL <br> | ||
+ | Insert = 7µL <br> | ||
+ | |||
+ | ==== NEB Enzymes ==== | ||
+ | |||
+ | For each samples add sterilized water to obtain a maximum volume equals to 8µL. <br> | ||
+ | |||
+ | Ligation mix (NEB): <br> | ||
+ | 3µL of T4 ligase + 3µL of T4 ligase buffer. <br> | ||
+ | Add 2µL / tube. <br> | ||
+ | Incubation 1h at RT. <br> | ||
+ | |||
+ | ==== Electroporation ==== | ||
+ | |||
+ | Electroporation cuvettes = 2mm ; inoculums of electrocompetent <i>E.coli</i> DH5alpha= 40µL; pulse = 2,5KVolt ; 1h of incubation. <br> | ||
+ | |||
+ | Spread 1mL of inoculums into a petri dish with LB + ampicillin (50mg/mL) (20/0,02mL). <br> |
Revision as of 03:27, 21 October 2009
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Contents |
Friday the 18th
Restriction digest
Ligation between B0014 and P1003
Restriction digest of BBa_B0014 (= 2,41µg/µL) by EcoRI and XbaI (3284bp):
DNA (10µg final) = 4,2µL
Buffer Eco R1 (NEB) = 5µL
H20 = 39,8µL
Eco R1 = 1µL
Incubation 1h at 37°C.
DNA purification with a nucleospin (macherey-nagel).
DNA = 50µL
Buffer 2 (NEB) = 6µL
BSA (NEB) = 0,6µL
H20 = 2,4µL
Xba 1 = 1µL
Incubation 1h at 37°C.
Restriction digest of P1003 (4,17µg/µL) by EcoRI and SpeI (967bp):
DNA (10µg final) = 2,4µL
Buffer Eco R1 (NEB) = 5µL
H20 = 40,6µL
BSA = 0,5µL
Eco R1 (NEB) = 1µL
Spe 1 (NEB) = 1µL
Incubation 1h at 37°C.
Ligation of B0014 with C0012
B0014 (= 2,41µg/µL) restriction digest by EcoRI and XbaI (3284bp):
Same samples as the restriction digest used for B0014 and P1003 ligation.
C0012 (1,56µg/µL) restriction digest by EcoRI and SpeI (1128bp):
DNA (10µg final) = 6,4µL
Buffer Eco R1 (NEB) = 5µL
H20 = 36,1µL
BSA = 0,5µL
Eco R1 (NEB) = 1µL
Spe 1 (NEB) = 1µL
Incubation 1h at 37°C.
DNA electrophoresis
85 Volt, 15 minutes.
105 Volt, 40 minutes.
Ladder fermentas 1 Kb.
Samples:
DNA purification
Kit Qiagen “gel extraction kit”, final volume = 50µL.
Ligation
Ligation schemes: plasmid / insert:
BBa_B0014/BBa_P1003 ;
BBa_B0014/BBa_C0012.
First report:
Plasmid = 1µL
Insert = 5µL
Second report:
Plasmid = 1µL
Insert = 3µL
Third report:
Plasmid = 1µL
Insert = 7µL
NEB Enzymes
For each samples add sterilized water to obtain a maximum volume equals to 8µL.
Ligation mix (NEB):
3µL of T4 ligase + 3µL of T4 ligase buffer.
Add 2µL / tube.
Incubation 1h at RT.
Electroporation
Electroporation cuvettes = 2mm ; inoculums of electrocompetent E.coli DH5alpha= 40µL; pulse = 2,5KVolt ; 1h of incubation.
Spread 1mL of inoculums into a petri dish with LB + ampicillin (50mg/mL) (20/0,02mL).