User:DavidC/2 October 2009

Ligation between BBa_B0030 and BBa_R0010
Restriction digest of R0010 by SpeI and PstI (2279bp):

DNA (miniprep) = 30µL Buffer H (TAKARA) =4µL H20 = 4µL F Spe1 (TAKARA) = 1µL Pst1 (TAKARA) = 1µL 1 hour of incubation at 37°C.

Restriction digest of BBa_B0030 by PstI and XbaI (15bp):

DNA (miniprep) = 30µL Buffer M (TAKARA) =4µL H20 = 4µL Pst1 (TAKARA) = 1µL Xba1 (TAKARA) = 1µL 1 hour of incubation at 37°C.

Ligation between BBa_B0014 and BBa_P1003 + BBa_B0014
Restriction digest of BBa_B0014 by EcoRI and SpeI (95bp):

DNA (miniprep) = 30µL Buffer M (TAKARA) =4µL H20 = 4µL EcoR1 (TAKARA) = 1µL Spe1 (TAKARA) = 1µL 1 hour of incubation at 37°C.

Restriction digst of BBa_P1003 + BBa_B0014 by EcoRI and XbaI (3770bp):

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
Restriction digest of COS by XbaI and SpeI (250bp):

DNA (miniprep) = 30µL Buffer M (TAKARA) =4µL H20 = 4µL Xba1 (TAKARA) = 1µL Spe1 (TAKARA) = 1µL 1 hour of incubation at 37°C.

Restriction digest of pSB1A2 by SpeI and XbaI (2079bp):

DNA (miniprep) = 30µL Buffer M (TAKARA) =4µL H20 = 4µL Spe1 (TAKARA) = 1µL Xba1 (TAKARA) = 1µL 1 hour of incubation at 37°C.

Ligation of the D protein (PrtD) with the adenovirus 5 penton base (ADV5pb)
Restriction digest of PrtD by SpeI (385bp):

DNA (PCR) = 5µL Buffer M (TAKARA) =2µL H20 = 12µL Spe1 (TAKARA) = 1µL 1 hour of incubation at 37°C.

Restriction digest of ADV5pb by XbaI (1715bp):

DNA (PCR) = 5µL Buffer M (TAKARA) =2µL BSA (TAKARA) = 2µL H20 = 10µL Xba1 (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_R0010, BBa_B0030, BBaB0014, BBa_P1003, adenovirus penton base with BioBrick prefix and suffix (ADV5pb BBa), D protein with BioBrick prefix and suffix (D prt BBa)



Interpretation:

We obtain all the fragments but we always have a problem of mistmacth for ADV5 pb primers, and the B0030 fragment is impossible to see into the photo, in fact there is a few DNA, and the fragment is short around 15bp.

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

Ligation
Ligation between COS and pSB1A2 plasmid backbone (TAKARA, DNA ligation kit) :

First report : COS = 3,5µL pSB1A2 = 0,5µL Solution A = 16µL Solution B = 4µL

Second report: COS = 6µL pSB1A2 = 1µL Solution A = 28µL Solution B = 7µL

1 hours of incubation at room temperature

Ligation between the D protein and ADV5pb (NEB enzyme):

First report : PrtD = 2,5µL ADV5pb = 5,5µL NEB T4 ligase buffer= 1µL NEB T4 ligase = 1µL

Second report:

PrtD = 3µL ADV5pb = 5µL NEB T4 ligase buffer= 1µL NEB T4 ligase = 1µL

Third report: PrtD = 4µL ADV5pb = 4µL NEB T4 ligase buffer= 1µL NEB T4 ligase = 1µL

Fourth report: PrtD = 2µL ADV5pb = 2µL NEB T4 ligase buffer= 1µL NEB T4 ligase = 1µL

1 hour at room temperature

Ligation between BBa_B0030 and BBa_R0010 (TAKARA, DNA ligation kit) :

First report : Plasmid (R0010) = 0,5µL Insert (B0030) = 5µL Solution A = 22µL Solution B = 5,5µL

Second report: Plasmid (R0010) = 0,5µL Insert (B0030) = 6µL Solution A = 26µL Solution B = 6,5µL

1 hour of incubation at room temperature.

Ligation between BBa_B0014 and BBa_P1003 + BBa_B0014 (TAKARA, DNA ligation kit) :

First report : Plasmid (P1003 + B0014) = 0,5µL Insert (B0014) = 5µL Solution A = 22µL Solution B = 5,5µL

Second report : Plasmid (P1003 + B0014) = 0,5µL Insert (B0014) = 6µL Solution A = 26µL Solution B = 6,5µL

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).