User:DavidC/9 September 2009

From 2009.igem.org

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=== 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. <br>
 +
 +
[[Image:W0909.png|center]]
 +
 +
<br>
 +
 +
==== 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>

Latest revision as of 02:44, 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

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.

W0909.png


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).

Retrieved from "http://2009.igem.org/User:DavidC/9_September_2009"