User:DavidC/9 October 2009

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(DNA electrophoresis)
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9 :00 pm: Cocktail party
9 :00 pm: Cocktail party
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===Experiments===
==== DNA extraction ====
==== DNA extraction ====

Revision as of 02:43, 22 October 2009

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Contents

Friday the 9th

Ethic debate

Issues of synthetic biology : state of art, state of mind

Programme

5:30 pm: Reception of the participants

6:00 pm: :Introduction of :

  • Synthetic biology, François Le Fèvre
  • The Double vectorisation system (DVS) project developed by the team

6:15 pm: Round-table leaded by Thierry Magnin and Ranya Jamali:

  • Synthetic Biology / DVS Project – Posing of the risks and benefits: what are the risks, can you avoid them, what are the effects on man, animal and environment, the advantages of this discipline, where does science stops and where does creation starts? Fears of populations...
  • Regulations, Access and law: to what extent must knowledge be protected, emphasizing the concept of "non-patentability" as well as regulations…

9 :00 pm: Cocktail party

Experiments

DNA extraction

Plasmid extraction by using minipreps (promega) on:
BBa_P1001 + BBaB0014;
BBa_P1003 + BBa_B0014;
BBa_B0030 + BBa_C0012 + BBa_B0014,
BBa_R0040;
BBa_C0040;
BBa_B0014;
BBa_B0030;
BBa_P1001.

Restriction digest

Ligation between BBa_P1001 and BBa_B0014

Restriction digest of BBa_B0014 by PstI and XbaI (95bp):

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

Restriction digest of BBa_P1001 by PstI and SpeI (5746bp):

DNA (miniprep) = 30µL
Buffer H (TAKARA) = 4µL
H20 = 4µL
PstI (TAKARA) = 1µL
SpeI (TAKARA) = 1µL
1 hour of incubation at 37°C.

Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030:

Restriction digest of BBa_B0030 by PstI and SpeI (2094bp):

DNA (miniprep) = 30µL
Buffer H (TAKARA) = 4µL
H20 = 4µL
PstI (TAKARA) = 1µL
SpeI (TAKARA) = 1µL
1 hour of incubation at 37°C.

Restriction digest of BBa_C0012 + BBa_B0014 by XbaI and PstI (1223bp):

DNA (miniprep) = 30µL
Buffer H (TAKARA) = 4µL
H20 = 4µL
PstI (TAKARA) = 1µL
XbaI (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.

F0910.png


Samples: BBa_R0040, BBa_B0030, BBa_C0012 + BBa_B0014.

F0910(2).png


DNA purification

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

Cell culture

Growth of E.coli DH5alpha into 5mL of LB medium with 5µL of the correct antibiotic (kanamycin = 50mg/mL, ampicillin = 50mg/mL and tetracycline = 12,5mg/mL)

Samples:
p 53 (3 times);
BBa_R0010;
BBa_B0030;
BBa_E0240;
BBa_B0014;

Ligation

Ligation between BBa_P1001 and BBa_B0014

First report:
Plasmid (P1001) = 2µL
Insert (B0014) = 6µL
Solution A = 3µL
Solution B = 2µL

Second report:
Plasmid (P1001) = 1µL
Insert (B0014) = 6µL
Solution A = 3µL
Solution B = 2µL

Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030

First report:
Plasmid (B0030) = 2µL
Insert (C0012 + B0014) = 0,5µL
Solution A = 5,5µL
Solution B = 2µL

Second report:
Plasmid (B0030) = 2µL
Insert (C0012 + B0014) = 1µL
Solution A = 6µL
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).