User:DavidC/10 October 2009

From 2009.igem.org

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=== Saturday the 10th ===
 +
 +
==== DNA extraction ===
 +
 +
Plasmid extraction by using minipreps (promega): <br>
 +
BBa_B0014; <br>
 +
BBa_E0240; <br>
 +
BBa_B0030; <br>
 +
BBa_R0010; <br>
 +
p53. <br>
 +
 +
==== Cell culture ====
 +
 +
Growth of <i>E.coli</i> DH5alpha into 500mL of LB medium with 500µL of ampicillin = 50mg/mL. <br><br>
 +
 +
Samples: <br>
 +
p53 (2 times); <br>
 +
 +
==== Restriction ====
 +
 +
==== Ligation between BBa_R0010 with BBa_B0030: ====
 +
 +
Restriction digest of BBa_B0030 by EcoRI and XbaI (2094bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) = 4µL <br>
 +
H20 = 4µL <br>
 +
EcoRI (TAKARA) = 1µL <br>
 +
XbaI (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br><br>
 +
 +
Restriction digest of BBa_R0010 by EcoRI and SpeI (200bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer EcoRI (NEB) = 13µL <br>
 +
H20 = 13µL <br>
 +
EcoRI (NEB) = 1µL <br>
 +
SpeI (NEB) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== Electrophoresis ====
 +
 +
==== DNA electrophoresis ====
 +
 +
85 Volt, 15 minutes. <br>
 +
105 Volt, 40 minutes. <br>
 +
Ladder fermentas 1 Kb. <br>
 +
 +
Samples: BBa_B0030, BBa_R0010.
 +
<br>
 +
 +
[[image:SA1010.png|center]]
 +
<br>
 +
 +
==== Ligation ====
 +
 +
==== Ligation between BBa_R0010 and BB_B0030 ====
 +
 +
First report: <br>
 +
Plasmid (B0030) = 2µL <br>
 +
Insert (R0010) = 8µL <br>
 +
Solution A = 12µL <br>
 +
Solution B = 3µL <br><br>
 +
 +
Second report: <br>
 +
Plasmid (B0030) = 2µL <br>
 +
Insert (R0010) = 6µL <br>
 +
Solution A = 6µL <br>
 +
Solution B = 2µL <br>
 +
 +
==== Ligation between BBa_P1001 and BBa_B0014 ====
 +
 +
First report: <br>
 +
Plasmid (P1001) = 2µL <br>
 +
Insert (B0014) = 10µL <br>
 +
Solution A = 12µL <br>
 +
Solution B = 4µL <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>
 +
 +
==== Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030: ====
 +
 +
First report: <br>
 +
Plasmid (C0012 + B0014) = 2µL <br>
 +
Insert (B0030) = 10µL <br>
 +
Solution A = 12µL <br>
 +
Solution B = 4µL <br>

Latest revision as of 17:27, 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

Saturday the 10th

= DNA extraction

Plasmid extraction by using minipreps (promega):
BBa_B0014;
BBa_E0240;
BBa_B0030;
BBa_R0010;
p53.

Cell culture

Growth of E.coli DH5alpha into 500mL of LB medium with 500µL of ampicillin = 50mg/mL.

Samples:
p53 (2 times);

Restriction

Ligation between BBa_R0010 with BBa_B0030:

Restriction digest of BBa_B0030 by EcoRI and XbaI (2094bp):

DNA (miniprep) = 30µL
Buffer M (TAKARA) = 4µL
H20 = 4µL
EcoRI (TAKARA) = 1µL
XbaI (TAKARA) = 1µL
1 hour of incubation at 37°C.

Restriction digest of BBa_R0010 by EcoRI and SpeI (200bp):

DNA (miniprep) = 30µL
Buffer EcoRI (NEB) = 13µL
H20 = 13µL
EcoRI (NEB) = 1µL
SpeI (NEB) = 1µL
1 hour of incubation at 37°C.

Electrophoresis

DNA electrophoresis

85 Volt, 15 minutes.
105 Volt, 40 minutes.
Ladder fermentas 1 Kb.

Samples: BBa_B0030, BBa_R0010.

SA1010.png


Ligation

Ligation between BBa_R0010 and BB_B0030

First report:
Plasmid (B0030) = 2µL
Insert (R0010) = 8µL
Solution A = 12µL
Solution B = 3µL

Second report:
Plasmid (B0030) = 2µL
Insert (R0010) = 6µL
Solution A = 6µL
Solution B = 2µL

Ligation between BBa_P1001 and BBa_B0014

First report:
Plasmid (P1001) = 2µL
Insert (B0014) = 10µL
Solution A = 12µL
Solution B = 4µL

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).

Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030:

First report:
Plasmid (C0012 + B0014) = 2µL
Insert (B0030) = 10µL
Solution A = 12µL
Solution B = 4µL

Retrieved from "http://2009.igem.org/User:DavidC/10_October_2009"