User:DavidC/7 October 2009

Trial for amplification of ligation between D protein and adenovirus 5 penton base
Sample: D protein (D prt) and adenovirus 5 penton base (ADV5 pb) digest by Spe I and Xba I (samples created on the 2nd of October).

Primers: Forward : Pref_ATG_Fw Reverse : Suffixe_Rv

DNA: 5µL of each samples: first, second, third and fourth reports.

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.

Samples: Fusion protein report 1, 2, 3, 4.



We obtain a fragment at 385bp, so the ligation does not work as expected. The fragment at 385bp is similar to the D protein, so there is not enough adenovirus 5 penton base into the sample or there is no DNA. We will change the next protocol and try to integrate the D protein and the ADV5 pb into plasmid vector independently and join them as a plasmid vector and a insert.

Restriction digest of adenovirus 5 penton base (1715bp), COS (250bp) and D protein (380bp) by Xba I and Pst I :
DNA (PCR) = 10µL Buffer M (TAKARA) = 2µL H20 = 6µL Xba I (TAKARA) = 1µL Pst I (TAKARA) = 1µL 1 hour of incubation at 37°C.

Restriction digest of pSB1A2 Xba I and Pst I (2079bp):
DNA (PCR) = 10µL Buffer M (TAKARA) = 2µL H20 = 6µL Xba I (TAKARA) = 1µL Pst 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: COS with Biobrick prefix and sufix (COS BBa)(250bp), D protein with Biobrick prefix and sufix (D prt BBa)(385bp), adenovirus penton base with Biobrick prefix and sufix (ADV5pb BBa)(1715bp), pSB1A2 (2079bp).



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

Ligation between ADV5 pb and pSB1A2:
First report: Plasmid (pSB1A2) = 2µL Insert (ADV5 pb) = 6µL Solution A = 1µL Solution B = 1µL

Second report: Plasmid (pSB1A2) = 3,5µL Insert (ADV5 pb) = 10µL Solution A = 2µL Solution B = 2µL

2 hours of incubation at room temperature.

Same protocol for: Ligation of COS with pSB1A2. Ligation of D protein with pSB1A2.