User:DavidC/17 September 2009

From 2009.igem.org

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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 03:25, 21 October 2009

framless



August
MTWTFSS
          [http://2009.igem.org/User:DavidC/1_August_2009 1] [http://2009.igem.org/User:DavidC/2_August_2009 2]
[http://2009.igem.org/User:DavidC/3_August_2009 3] [http://2009.igem.org/User:DavidC/4_August_2009 4] [http://2009.igem.org/User:DavidC/5_August_2009 5] [http://2009.igem.org/User:DavidC/6_August_2009 6] [http://2009.igem.org/User:DavidC/7_August_2009 7] [http://2009.igem.org/User:DavidC/8_August_2009 8] [http://2009.igem.org/User:DavidC/9_August_2009 9]
[http://2009.igem.org/User:DavidC/10_August_2009 10] [http://2009.igem.org/User:DavidC/11_August_2009 11] [http://2009.igem.org/User:DavidC/12_August_2009 12] [http://2009.igem.org/User:DavidC/13_August_2009 13] [http://2009.igem.org/User:DavidC/14_August_2009 14] [http://2009.igem.org/User:DavidC/15_August_2009 15] [http://2009.igem.org/User:DavidC/16_August_2009 16]
[http://2009.igem.org/User:DavidC/17_August_2009 17] [http://2009.igem.org/User:DavidC/18_August_2009 18] [http://2009.igem.org/User:DavidC/19_August_2009 19] [http://2009.igem.org/User:DavidC/20_August_2009 20] [http://2009.igem.org/User:DavidC/21_August_2009 21] [http://2009.igem.org/User:DavidC/22_August_2009 22] [http://2009.igem.org/User:DavidC/23_August_2009 23]
[http://2009.igem.org/User:DavidC/24_August_2009 24] [http://2009.igem.org/User:DavidC/25_August_2009 25] [http://2009.igem.org/User:DavidC/26_August_2009 26] [http://2009.igem.org/User:DavidC/27_August_2009 27] [http://2009.igem.org/User:DavidC/28_August_2009 28] [http://2009.igem.org/User:DavidC/29_August_2009 29] [http://2009.igem.org/User:DavidC/30_August_2009 30]
[http://2009.igem.org/User:DavidC/31_August_2009 31]
September
MTWTFSS
  [http://2009.igem.org/User:DavidC/1_September_2009 1] [http://2009.igem.org/User:DavidC/2_September_2009 2] [http://2009.igem.org/User:DavidC/3_September_2009 3] [http://2009.igem.org/User:DavidC/4_September_2009 4] [http://2009.igem.org/User:DavidC/5_September_2009 5] [http://2009.igem.org/User:DavidC/6_September_2009 6]
[http://2009.igem.org/User:DavidC/7_September_2009 7] [http://2009.igem.org/User:DavidC/8_September_2009 8] [http://2009.igem.org/User:DavidC/9_September_2009 9] [http://2009.igem.org/User:DavidC/10_September_2009 10] [http://2009.igem.org/User:DavidC/11_September_2009 11] [http://2009.igem.org/User:DavidC/12_September_2009 12] [http://2009.igem.org/User:DavidC/13_September_2009 13]
[http://2009.igem.org/User:DavidC/14_September_2009 14] [http://2009.igem.org/User:DavidC/15_September_2009 15] [http://2009.igem.org/User:DavidC/16_September_2009 16] [http://2009.igem.org/User:DavidC/17_September_2009 17] [http://2009.igem.org/User:DavidC/18_September_2009 18] [http://2009.igem.org/User:DavidC/19_September_2009 19] [http://2009.igem.org/User:DavidC/20_September_2009 20]
[http://2009.igem.org/User:DavidC/21_September_2009 21] [http://2009.igem.org/User:DavidC/22_September_2009 22] [http://2009.igem.org/User:DavidC/23_September_2009 23] [http://2009.igem.org/User:DavidC/24_September_2009 24] [http://2009.igem.org/User:DavidC/25_September_2009 25] [http://2009.igem.org/User:DavidC/26_September_2009 26] [http://2009.igem.org/User:DavidC/27_September_2009 27]
[http://2009.igem.org/User:DavidC/28_September_2009 28] [http://2009.igem.org/User:DavidC/29_September_2009 29] [http://2009.igem.org/User:DavidC/30_September_2009 30]
October
MTWTFSS
      [http://2009.igem.org/User:DavidC/1_October_2009 1] [http://2009.igem.org/User:DavidC/2_October_2009 2] [http://2009.igem.org/User:DavidC/3_October_2009 3] [http://2009.igem.org/User:DavidC/4_October_2009 4]
[http://2009.igem.org/User:DavidC/5_October_2009 5] [http://2009.igem.org/User:DavidC/6_October_2009 6] [http://2009.igem.org/User:DavidC/7_October_2009 7] [http://2009.igem.org/User:DavidC/8_October_2009 8] [http://2009.igem.org/User:DavidC/9_October_2009 9] [http://2009.igem.org/User:DavidC/10_October_2009 10] [http://2009.igem.org/User:DavidC/11_October_2009 11]
[http://2009.igem.org/User:DavidC/12_October_2009 12] [http://2009.igem.org/User:DavidC/13_October_2009 13] [http://2009.igem.org/User:DavidC/14_October_2009 14] [http://2009.igem.org/User:DavidC/15_October_2009 15] [http://2009.igem.org/User:DavidC/16_October_2009 16] [http://2009.igem.org/User:DavidC/17_October_2009 17] [http://2009.igem.org/User:DavidC/18_October_2009 18]
[http://2009.igem.org/User:DavidC/19_October_2009 19] [http://2009.igem.org/User:DavidC/20_October_2009 20] [http://2009.igem.org/User:DavidC/21_October_2009 21] [http://2009.igem.org/User:DavidC/22_October_2009 22] [http://2009.igem.org/User:DavidC/23_October_2009 23] [http://2009.igem.org/User:DavidC/24_October_2009 24] [http://2009.igem.org/User:DavidC/25_October_2009 25]
[http://2009.igem.org/User:DavidC/26_October_2009 26] [http://2009.igem.org/User:DavidC/27_October_2009 27] [http://2009.igem.org/User:DavidC/28_October_2009 28] [http://2009.igem.org/User:DavidC/29_October_2009 29] [http://2009.igem.org/User:DavidC/30_October_2009 30] [http://2009.igem.org/User:DavidC/31_October_2009 31]


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).

Retrieved from "http://2009.igem.org/User:DavidC/17_September_2009"