User:DavidC/1 October 2009

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 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).