User:DavidC/14 September 2009

From 2009.igem.org

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August
MTWTFSS
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31
September
MTWTFSS
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14 15 16 17 18 19 20
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28 29 30
October
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5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
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Contents

Monday the 14th

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).

PCR

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

M1409.png


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.