User:DavidC/18 September 2009

From 2009.igem.org

framless



August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31


Contents

Friday the 18th

Restriction digest

Ligation between BBa_B0014 and BBa_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 BBa_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 beween BBa_B0014 and BBa_C0012

BBa_B0014 (= 2,41µg/µL) restriction digest by EcoRI and XbaI (3284bp):

Same samples as the restriction digest used for B0014 and P1003 ligation.

BBa_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: BBa_P1003, BBa_B0014, BBa_C0012.

F1809.png


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).