User:DavidC/14 September 2009

Ligation: samples digested on the 13th of September
Ligation pairs: plasmid / insert :

BBa_B0014 / BBa_C0012

First report: Plasmid = 2µL Insert = 5µL

Second report: Plasmid = 1,5µL Insert = 5µL

Third report: Plasmide = 1µL Insert = 7µL

NEB Enzymes
For each samples add sterilized water to obtain a maximum volume equals to 8µL. Ligation mix (NEB): 1,5µL T4 ligase + 1,5µL T4 ligase buffer. Add 2µL / tube. Incubation 1h at RT.

TAKARA DNA ligation kit
First report: Solution A (Buffer) = 28µL Solution B (Enzyme) = 7µL Incubation 1h at 16°C.

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).

D protein
Primers: Forward : Préfixe ATG BBa Reverse : Prt D_Rv_Bal1

ADN : 1. Lambda bacteriophage; 2. Purified and amplified D protein.

Cycles: 94°C 2minutes; (94°C 1 minute; 46°C 1 minutes; 72°C 1 minute(s)) X 35; 72°C 4 minutes;

Penton base from the adenovirus 5
Primers: Forward : ADV5Fw_Bal1 Reverse : ADV5Rv_Bal1

ADN : Purified and amplified penton base from plasmid coding for adenovirus 5.

Cycles: 94°C 2minutes; (94°C 1 minute; 57,3°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: Adenovirus penton base, D protein



Interpretation:

We obtain a small amplification for D protein on the sample 4. But other samples have bad results, we obtain to much DNA for samples 1 and 2 PCR, we also amplified the wrong DNA, maybe we use a bad temperature for primers annealing, finally sample 3 is negative.