User:DavidC/14 September 2009

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=== Monday the 14th ===
 +
 +
==== Ligation: samples digested on the 13th of September ====
 +
 +
Ligation pairs: plasmid / insert : <br>
 +
 +
BBa_B0014 / BBa_C0012 <br>
 +
 +
First report: <br>
 +
Plasmid = 2µL <br>
 +
Insert = 5µL <br>
 +
 +
Second report: <br>
 +
Plasmid = 1,5µL <br>
 +
Insert = 5µL <br>
 +
 +
Third report: <br>
 +
Plasmide = 1µL <br>
 +
Insert = 7µL <br>
 +
 +
==== NEB Enzymes ====
 +
 +
For each samples add sterilized water to obtain a maximum volume equals to 8µL. <br>
 +
Ligation mix (NEB): <br>
 +
1,5µL T4 ligase + 1,5µL T4 ligase buffer. <br>
 +
Add 2µL / tube. <br>
 +
Incubation 1h at RT. <br>
 +
 +
==== TAKARA DNA ligation kit ===
 +
 +
First report:
 +
Solution A (Buffer) = 28µL <br>
 +
Solution B (Enzyme) = 7µL <br>
 +
Incubation 1h at 16°C. <br>
 +
 +
==== Electroporation ====
 +
 +
Electroporation cuvettes = 2mm ; inoculums of electrocompetent <i>E.coli</i> DH5alpha= 40µL; pulse = 2,5KVolt ; 1h of incubation. <br>
 +
 +
Spread 1mL of inoculums into a petri dish with LB + ampicillin (50mg/mL) (20/0,02mL). <br>
 +
 +
==== PCR ====
 +
 +
==== D protein ====
 +
 +
Primers: <br>
 +
Forward : Préfixe ATG BBa <br>
 +
Reverse : Prt D_Rv_Bal1 <br><br>
 +
 +
ADN : <br>
 +
1. Lambda bacteriophage; <br>
 +
2. Purified and amplified D protein. <br><br>
 +
 +
Cycles: <br>
 +
94°C 2minutes; <br>
 +
(94°C 1 minute; <br>
 +
46°C 1 minutes; <br>
 +
72°C 1 minute(s)) X 35; <br>
 +
72°C 4 minutes; <br>
 +
 +
==== Penton base from the adenovirus 5 ====
 +
 +
Primers: <br>
 +
Forward : ADV5Fw_Bal1 <br>
 +
Reverse : ADV5Rv_Bal1 <br><br>
 +
 +
ADN : <br>
 +
Purified and amplified penton base from plasmid coding for adenovirus 5. <br><br>
 +
 +
Cycles: <br>
 +
94°C 2minutes; <br>
 +
(94°C 1 minute; <br>
 +
57,3°C 1 minutes; <br>
 +
72°C 2 minute(s)) X 35; <br>
 +
72°C 5 minutes; <br>
 +
 +
==== DNA electrophoresis ====
 +
 +
85 Volt, 15 minutes. <br>
 +
105 Volt, 40 minutes. <br>
 +
Ladder fermentas 1 Kb. <br><br>
 +
 +
Samples: Adenovirus penton base, D protein <br>
 +
 +
[[image:M1409.png|center]]
 +
 +
 +
Interpretation: <br>
 +
 +
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. <br>

Latest revision as of 14:39, 21 October 2009

framless



August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31


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.

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