User:DavidC/9 September 2009

From 2009.igem.org

(Difference between revisions)
(New page: {{Template:Supbiotechcss6.css}} {{Template:SupbiotechparisEn}} <center> <div style="color: black; margin-bottom: 30px; width: 500px;"> {|align="center" ||{{#calendar: title=User:DavidC |...)
Line 12: Line 12:
</div>
</div>
</center>
</center>
 +
 +
 +
=== Wednesday the 9th ===
 +
 +
==== Restriction digest: ====
 +
 +
==== Ligation between BBa_P1002 and BBa_B0014: ====
 +
 +
BBa_B0014 (concentration = 3,70µg/µL) restriction digest by EcoRI and XbaI (3284pb): <br>
 +
 +
DNA (10µg final) = 2,7µL <br>
 +
Buffer Eco R1 (NEB) = 5µL <br>
 +
H20 = 41,3µL <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>
 +
 +
BBa_P1002 (concentration = 2,86µg/µL) restriction digest by EcoRI and SpeI (943bp): <br>
 +
 +
DNA (10µg final) = 3,5µL <br>
 +
Buffer Eco R1 (NEB) = 5µL <br>
 +
BSA = 0,5µL <br>
 +
H20 = 39µL <br>
 +
Eco R1 (NEB) = 1µL <br>
 +
Spe 1 (NEB) = 1µL <br>
 +
Incubation 1h at 37°C. <br>
 +
 +
==== Ligation between BBa_C0012 and BBa_B0014: ====
 +
 +
BBa_B0014 (concentration = 3,70µg/µL)restriction digest by EcoRI and XbaI (3284bp): <br>
 +
 +
Same sample use for ligation between P1002 and B0014. <br>
 +
 +
BBa_C0012 (concentration = 1,49µg/µL) restriction digest by EcoRI and SpeI (1128bp): <br>
 +
 +
DNA (10µg final) = 6,7µL <br>
 +
Buffer Eco R1 (NEB) = 5µL <br>
 +
H20 = 35,8µL <br>
 +
BSA = 0,5µL <br>
 +
Eco R1 (NEB) = 1µL <br>
 +
Spe 1 (NEB) = 1µL <br>
 +
Incubation 1h at 37°C. <br>
 +
 +
==== Restriction digest of BBa_C0040 (concentration = 4,35µg/µL) by EcoRI and SpeI (660bp): ====
 +
 +
DNA (10µg final)= 2,3µL <br>
 +
Buffer Eco R1 (NEB) = 5µL <br>
 +
H20 = 40,7µL <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: BBa_C0012, BBa_C0040, BBa_B0014, BBa_P1002.
 +
 +
[[W0909.png|center]]
 +
 +
 +
==== Ligation ====
 +
 +
Ligation pairs, plasmid / insert (respectively): <br>
 +
 +
BBa_B0014/ BBa_C0012 <br>
 +
BBa_B0014/BBa_P1002 <br>
 +
 +
First ratio: <br>
 +
Plasmid = 1µL <br>
 +
Insert = 4µL <br><br>
 +
 +
Second ratio: <br>
 +
Plasmid = 1µL <br>
 +
Insert = 2µL <br><br>
 +
 +
Third ratio: <br>
 +
Plasmid = 2µL <br>
 +
Insert = 4µL <br><br>
 +
 +
Fourth ratio: <br>
 +
Plasmid = 1µL <br>
 +
Insert = 5µL <br><br>
 +
 +
Add sterilized water to maximum volume equal to 8µL. <br>
 +
 +
Ligation mix : <br>
 +
9µL T4 ligase + 9µL T4 ligase buffer. <br>
 +
Add 2µL / tube. <br>
 +
Incubate 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 02:41, 21 October 2009

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

Wednesday the 9th

Restriction digest:

Ligation between BBa_P1002 and BBa_B0014:

BBa_B0014 (concentration = 3,70µg/µL) restriction digest by EcoRI and XbaI (3284pb):

DNA (10µg final) = 2,7µL
Buffer Eco R1 (NEB) = 5µL
H20 = 41,3µL
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.

BBa_P1002 (concentration = 2,86µg/µL) restriction digest by EcoRI and SpeI (943bp):

DNA (10µg final) = 3,5µL
Buffer Eco R1 (NEB) = 5µL
BSA = 0,5µL
H20 = 39µL
Eco R1 (NEB) = 1µL
Spe 1 (NEB) = 1µL
Incubation 1h at 37°C.

Ligation between BBa_C0012 and BBa_B0014:

BBa_B0014 (concentration = 3,70µg/µL)restriction digest by EcoRI and XbaI (3284bp):

Same sample use for ligation between P1002 and B0014.

BBa_C0012 (concentration = 1,49µg/µL) restriction digest by EcoRI and SpeI (1128bp):

DNA (10µg final) = 6,7µL
Buffer Eco R1 (NEB) = 5µL
H20 = 35,8µL
BSA = 0,5µL
Eco R1 (NEB) = 1µL
Spe 1 (NEB) = 1µL
Incubation 1h at 37°C.

Restriction digest of BBa_C0040 (concentration = 4,35µg/µL) by EcoRI and SpeI (660bp):

DNA (10µg final)= 2,3µL
Buffer Eco R1 (NEB) = 5µL
H20 = 40,7µL
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_C0012, BBa_C0040, BBa_B0014, BBa_P1002.

center


Ligation

Ligation pairs, plasmid / insert (respectively):

BBa_B0014/ BBa_C0012
BBa_B0014/BBa_P1002

First ratio:
Plasmid = 1µL
Insert = 4µL

Second ratio:
Plasmid = 1µL
Insert = 2µL

Third ratio:
Plasmid = 2µL
Insert = 4µL

Fourth ratio:
Plasmid = 1µL
Insert = 5µL

Add sterilized water to maximum volume equal to 8µL.

Ligation mix :
9µL T4 ligase + 9µL T4 ligase buffer.
Add 2µL / tube.
Incubate 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).