User:DavidC/6 October 2009

From 2009.igem.org

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=== Tuesday the 6th ===
 +
 +
==== DNA extraction ====
 +
 +
Miniprep extraction (promega) of: <br>
 +
 +
BBa_B0014; <br>
 +
BBa_B0030; <br>
 +
BBa_P1001; <br>
 +
BBa_C0040; <br>
 +
BBa_R0040. <br>
 +
 +
==== Restriction digest ====
 +
 +
==== Ligation between BBa_B0014 and BBa_P1001: ====
 +
 +
Restriction digest of BBa_B0014 by PstI and SpeI (3284pb): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer (NEB) = 5µL <br>
 +
H20 = 13µL <br>
 +
PstI (NEB) = 1µL <br>
 +
Spe I (NEB) = 1µL <br>
 +
1 hour of incubation at 37°C. <br><br>
 +
 +
Restriction digest of BBa_P1001 by PstI and XbaI (1279bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) = 4µL <br>
 +
H20 = 4µL <br>
 +
Eco RI (TAKARA) = 1µL <br>
 +
Spe I (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030 ====
 +
 +
Restriction digest of BBa_C0012 + BBa_B0014 by EcoRI and SpeI (1223bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer (NEB) = 5µL <br>
 +
H20 = 13µL <br>
 +
PstI (NEB) = 1µL <br>
 +
Spe I (NEB) = 1µL <br>
 +
1 hour of incubation at 37°C. <br><br>
 +
 +
Restriction digest of BBa_B0030 by EcoRI and XbaI (2094bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) = 4µL <br>
 +
H20 = 4µL <br>
 +
Eco RI (TAKARA) = 1µL <br>
 +
Spe I (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== Restriction digest of BBa_C0040 by Eco RI and Spe I (660bp) ====
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) = 4µL <br>
 +
H20 = 4µL <br>
 +
Eco RI (TAKARA) = 1µL <br>
 +
Spe I (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== Restriction of BBa_R0040 by Eco RI and Xba I (2133bp) ====
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) = 4µL <br>
 +
H20 = 4µL <br>
 +
Eco RI (TAKARA) = 1µL <br>
 +
Xba I (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== DNA electrophoresis ====
 +
 +
85 Volt, 15 minutes. <br>
 +
105 Volt, 40 minutes. <br>
 +
Ladder fermentas 1 Kb. <br>
 +
 +
 +
Samples: BBa_P1001, BBa_B0014, BBa_C0040, BBa_R0040, BBa_B0030, BBa_C0012 + BBa_B0014.
 +
<br>
 +
 +
[[image:TU0610.png|center]]
 +
<br>
 +
 +
==== DNA purification ====
 +
 +
Kit Qiagen “gel extraction kit”, final volume = 50µL. <br>
 +
 +
==== Ligation ====
 +
 +
Ligation of BBa_B0014 with BBa_P1001 (TAKARA, DNA ligation kit) : <br>
 +
 +
First report: <br>
 +
Plasmid (P1001) = 1µL <br>
 +
Insert (B0014) = 7µL <br>
 +
Solution A = 1µL <br>
 +
Solution B = 1µL <br>
 +
 +
Second report: <br>
 +
Plasmid (P1001) = 0,5µL <br>
 +
Insert (B0014) = 4µL <br>
 +
Solution A = 4,5µL <br>
 +
Solution B = 1µL <br>
 +
 +
Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030 (TAKARA, DNA ligation kit) : <br>
 +
 +
First report: <br>
 +
Plasmid (B0030) = 1µL <br>
 +
Insert (C0012 + B0014) = 7µL <br>
 +
Solution A = 1µL <br>
 +
Solution B = 1µL <br>
 +
 +
Second report: <br>
 +
Plasmid (B0030) = 0,5µL <br>
 +
Insert (C0012 + B0014) = 4µL <br>
 +
Solution A = 4,5µL <br>
 +
Solution B = 1µL <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 16:13, 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

Tuesday the 6th

DNA extraction

Miniprep extraction (promega) of:

BBa_B0014;
BBa_B0030;
BBa_P1001;
BBa_C0040;
BBa_R0040.

Restriction digest

Ligation between BBa_B0014 and BBa_P1001:

Restriction digest of BBa_B0014 by PstI and SpeI (3284pb):

DNA (miniprep) = 30µL
Buffer (NEB) = 5µL
H20 = 13µL
PstI (NEB) = 1µL
Spe I (NEB) = 1µL
1 hour of incubation at 37°C.

Restriction digest of BBa_P1001 by PstI and XbaI (1279bp):

DNA (miniprep) = 30µL
Buffer M (TAKARA) = 4µL
H20 = 4µL
Eco RI (TAKARA) = 1µL
Spe I (TAKARA) = 1µL
1 hour of incubation at 37°C.

Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030

Restriction digest of BBa_C0012 + BBa_B0014 by EcoRI and SpeI (1223bp):

DNA (miniprep) = 30µL
Buffer (NEB) = 5µL
H20 = 13µL
PstI (NEB) = 1µL
Spe I (NEB) = 1µL
1 hour of incubation at 37°C.

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

DNA (miniprep) = 30µL
Buffer M (TAKARA) = 4µL
H20 = 4µL
Eco RI (TAKARA) = 1µL
Spe I (TAKARA) = 1µL
1 hour of incubation at 37°C.

Restriction digest of BBa_C0040 by Eco RI and Spe I (660bp)

DNA (miniprep) = 30µL
Buffer M (TAKARA) = 4µL
H20 = 4µL
Eco RI (TAKARA) = 1µL
Spe I (TAKARA) = 1µL
1 hour of incubation at 37°C.

Restriction of BBa_R0040 by Eco RI and Xba I (2133bp)

DNA (miniprep) = 30µL
Buffer M (TAKARA) = 4µL
H20 = 4µL
Eco RI (TAKARA) = 1µL
Xba I (TAKARA) = 1µL
1 hour of incubation at 37°C.

DNA electrophoresis

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


Samples: BBa_P1001, BBa_B0014, BBa_C0040, BBa_R0040, BBa_B0030, BBa_C0012 + BBa_B0014.

TU0610.png


DNA purification

Kit Qiagen “gel extraction kit”, final volume = 50µL.

Ligation

Ligation of BBa_B0014 with BBa_P1001 (TAKARA, DNA ligation kit) :

First report:
Plasmid (P1001) = 1µL
Insert (B0014) = 7µL
Solution A = 1µL
Solution B = 1µL

Second report:
Plasmid (P1001) = 0,5µL
Insert (B0014) = 4µL
Solution A = 4,5µL
Solution B = 1µL

Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030 (TAKARA, DNA ligation kit) :

First report:
Plasmid (B0030) = 1µL
Insert (C0012 + B0014) = 7µL
Solution A = 1µL
Solution B = 1µL

Second report:
Plasmid (B0030) = 0,5µL
Insert (C0012 + B0014) = 4µL
Solution A = 4,5µL
Solution B = 1µL

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/6_October_2009"