User:DavidC/7 October 2009

From 2009.igem.org

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

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: Fusion protein report 1, 2, 3, 4.

W0710(2).png


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: COS with Biobrick prefix and sufix (COS BBa)(250bp), D protein with Biobrick prefix and sufix (D prt BBa)(385bp), adenovirus penton base with Biobrick prefix and sufix (ADV5pb BBa)(1715bp), pSB1A2 (2079bp).

W0710.png


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.