User:DavidC/7 October 2009

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=== Wednesday the 7th ===
 +
 +
==== PCR ====
 +
 +
==== Trial for amplification of ligation between D protein and adenovirus 5 penton base ====
 +
 +
Sample: <br>
 +
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: <br>
 +
Forward : Pref_ATG_Fw <br>
 +
Reverse : Suffixe_Rv <br><br>
 +
 +
DNA: <br>
 +
5µL of each samples: first, second, third and fourth reports. <br>
 +
 +
Cycles: <br>
 +
94°C 2minutes; <br>
 +
(94°C 1 minute; <br>
 +
59°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>
 +
 +
 +
Samples:
 +
 +
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. <br><br>
 +
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. <br>
 +
 +
==== Restriction digest ====
 +
 +
==== Restriction digest of adenovirus 5 penton base (1715bp), COS (250bp) and D protein (380bp) by Xba I and Pst I : ====
 +
 +
DNA (PCR) = 10µL <br>
 +
Buffer M (TAKARA) = 2µL <br>
 +
H20 = 6µL <br>
 +
Xba I (TAKARA) = 1µL <br>
 +
Pst I (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== Restriction digest of pSB1A2 Xba I and Pst I (2079bp): ====
 +
 +
DNA (PCR) = 10µL <br>
 +
Buffer M (TAKARA) = 2µL <br>
 +
H20 = 6µL <br>
 +
Xba I (TAKARA) = 1µL <br>
 +
Pst I (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== DNA electrophoresis ====
 +
 +
85 Volt, 15 minutes. <br>
 +
105 Volt, 40 minutes. <br>
 +
Ladder fermentas 1 Kb. <br>
 +
 +
 +
 +
Samples:
 +
 +
==== DNA purification ====
 +
 +
Kit Qiagen “gel extraction kit”, final volume = 50µL. <br>
 +
 +
==== Ligation ====
 +
 +
==== Ligation between ADV5 pb and pSB1A2: ====
 +
 +
First report: <br>
 +
Plasmid (pSB1A2) = 2µL <br>
 +
Insert (ADV5 pb) = 6µL <br>
 +
Solution A = 1µL <br>
 +
Solution B = 1µL <br><br>
 +
 +
Second report: <br>
 +
Plasmid (pSB1A2) = 3,5µL <br>
 +
Insert (ADV5 pb) = 10µL <br>
 +
Solution A = 2µL <br>
 +
Solution B = 2µL <br><br>
 +
 +
2 hours of incubation at room temperature. <br><br>
 +
 +
Same protocol for: <br>
 +
Ligation of COS with pSB1A2. <br>
 +
Ligation of D protein with pSB1A2. <br>

Revision as of 04:18, 21 October 2009

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Contents

Wednesday the 7th

PCR

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:

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

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:

DNA purification

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

Ligation

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.