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User:DavidC/1 October 2009
From 2009.igem.org
Introduction Current therapies Gene therapies DVS Concept Tissue Vector Cell Vector Therapeutic plasmid Conclusion Implementation Tissue targeting Antitumor action Treatment modeling Conclusion
BioBricks Material & Method Bibliography
Thursday the 1st
PCR
D protein of Lambda phage
Primers :
Forward : Pref_ATG_Fw
Reverse : Suffixe_Rv
DNA :
Purified and amplified D protein from Lambda phage.
Cycles:
94°C 2minutes;
(94°C 1 minute;
59°C 1 minutes;
72°C 1 minute(s)) X 35;
72°C 4 minutes;
Adénovirus 5 penton base
Primers:
Forward : Pref_ATG_Fw
Reverse : Suffixe_Rv
DNA :
Purified and amplified penton base from the adenovirus 5.
Cycles:
94°C 2minutes;
(94°C 1 minute;
59°C 1 minutes;
72°C 2 minute(s)) X 35;
72°C 5 minutes;
Ligation of D protein and adenovirus 5 penton base
Restriction digest of samples by SpeI and XbaI.
Primers:
Forward : Pref_ATG_Fw
Reverse : Suffixe_Rv
DNA:
1. D protein restriction digests by SpeI and Adenovirus 5 penton base digests by XbaI.
2. D protein and Adenovirus 5 penton base digest by BalI.
Cycles:
94°C 2minutes;
(94°C 1 minute;
59°C 1 minutes;
72°C 2 minute(s)) X 35;
72°C 5 minutes;
DNA electrophoresis
85 Volt, 15 minutes.
105 Volt, 40 minutes.
Ladder fermentas 1 Kb.
DNA purification
No purification, We obtain negative results.
DNA extraction
Miniprep (promega):
Samples:
BBa_C0012 + BBa_B0014 ;
BBa_C0012.
Restriction digest
BBa_C0012 + BBa_B0014 digest by EcoRI and and XbaI (3302bp):
DNA (miniprep) = 30µL
Buffer M (TAKARA) =4µL
H20 = 4µL
EcoR1 (TAKARA) = 1µL
Xba1 (TAKARA) = 1µL
1 hour of incubation at 37°C.
Ligation between COS and pSB1A2 backbone
COS insert (purified PCR product) digests by XbaI and PstI (250bp):
DNA (miniprep) = 30µL
Buffer M (TAKARA) =4µL
H20 = 4µL
Xba1 (TAKARA) = 1µL
Pst1 (TAKARA) = 1µL
1 hour of incubation at 37°C.
pSB1A2 digests by XbaI and PstI (2079bp):
DNA (miniprep) = 30µL
Buffer M (TAKARA) =4µL
H20 = 4µL
Xba1 (TAKARA) = 1µL
Pst1 (TAKARA) = 1µL
1 hour of incubation 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 between COS and pSB1A2:
First report :
COS = 4µL
pSB1A2 = 1µL
Solution A = 20µL
Solution B = 5µL
Second report :
COS = 2µL
pSB1A2 = 0,5µL
Solution A = 10µL
Solution B = 2,5µL
Third report :
COS = 6µL
pSB1A2 = 1µL
Solution A = 24µL
Solution B = 6µL
1 hour of incubation at room temperature
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).
