User:DavidC/7 October 2009

From 2009.igem.org

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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: Fusion protein report 1, 2, 3, 4.
 +
<br>
 +
 +
[[image:W0710(2).png|center]]
 +
<br>
 +
 +
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: 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).
 +
<br>
 +
 +
[[image:W0710.png|center]]
 +
<br>
 +
 +
==== 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>

Latest revision as of 16:31, 21 October 2009

framless



August
MTWTFSS
          [http://2009.igem.org/User:DavidC/1_August_2009 1] [http://2009.igem.org/User:DavidC/2_August_2009 2]
[http://2009.igem.org/User:DavidC/3_August_2009 3] [http://2009.igem.org/User:DavidC/4_August_2009 4] [http://2009.igem.org/User:DavidC/5_August_2009 5] [http://2009.igem.org/User:DavidC/6_August_2009 6] [http://2009.igem.org/User:DavidC/7_August_2009 7] [http://2009.igem.org/User:DavidC/8_August_2009 8] [http://2009.igem.org/User:DavidC/9_August_2009 9]
[http://2009.igem.org/User:DavidC/10_August_2009 10] [http://2009.igem.org/User:DavidC/11_August_2009 11] [http://2009.igem.org/User:DavidC/12_August_2009 12] [http://2009.igem.org/User:DavidC/13_August_2009 13] [http://2009.igem.org/User:DavidC/14_August_2009 14] [http://2009.igem.org/User:DavidC/15_August_2009 15] [http://2009.igem.org/User:DavidC/16_August_2009 16]
[http://2009.igem.org/User:DavidC/17_August_2009 17] [http://2009.igem.org/User:DavidC/18_August_2009 18] [http://2009.igem.org/User:DavidC/19_August_2009 19] [http://2009.igem.org/User:DavidC/20_August_2009 20] [http://2009.igem.org/User:DavidC/21_August_2009 21] [http://2009.igem.org/User:DavidC/22_August_2009 22] [http://2009.igem.org/User:DavidC/23_August_2009 23]
[http://2009.igem.org/User:DavidC/24_August_2009 24] [http://2009.igem.org/User:DavidC/25_August_2009 25] [http://2009.igem.org/User:DavidC/26_August_2009 26] [http://2009.igem.org/User:DavidC/27_August_2009 27] [http://2009.igem.org/User:DavidC/28_August_2009 28] [http://2009.igem.org/User:DavidC/29_August_2009 29] [http://2009.igem.org/User:DavidC/30_August_2009 30]
[http://2009.igem.org/User:DavidC/31_August_2009 31]
September
MTWTFSS
  [http://2009.igem.org/User:DavidC/1_September_2009 1] [http://2009.igem.org/User:DavidC/2_September_2009 2] [http://2009.igem.org/User:DavidC/3_September_2009 3] [http://2009.igem.org/User:DavidC/4_September_2009 4] [http://2009.igem.org/User:DavidC/5_September_2009 5] [http://2009.igem.org/User:DavidC/6_September_2009 6]
[http://2009.igem.org/User:DavidC/7_September_2009 7] [http://2009.igem.org/User:DavidC/8_September_2009 8] [http://2009.igem.org/User:DavidC/9_September_2009 9] [http://2009.igem.org/User:DavidC/10_September_2009 10] [http://2009.igem.org/User:DavidC/11_September_2009 11] [http://2009.igem.org/User:DavidC/12_September_2009 12] [http://2009.igem.org/User:DavidC/13_September_2009 13]
[http://2009.igem.org/User:DavidC/14_September_2009 14] [http://2009.igem.org/User:DavidC/15_September_2009 15] [http://2009.igem.org/User:DavidC/16_September_2009 16] [http://2009.igem.org/User:DavidC/17_September_2009 17] [http://2009.igem.org/User:DavidC/18_September_2009 18] [http://2009.igem.org/User:DavidC/19_September_2009 19] [http://2009.igem.org/User:DavidC/20_September_2009 20]
[http://2009.igem.org/User:DavidC/21_September_2009 21] [http://2009.igem.org/User:DavidC/22_September_2009 22] [http://2009.igem.org/User:DavidC/23_September_2009 23] [http://2009.igem.org/User:DavidC/24_September_2009 24] [http://2009.igem.org/User:DavidC/25_September_2009 25] [http://2009.igem.org/User:DavidC/26_September_2009 26] [http://2009.igem.org/User:DavidC/27_September_2009 27]
[http://2009.igem.org/User:DavidC/28_September_2009 28] [http://2009.igem.org/User:DavidC/29_September_2009 29] [http://2009.igem.org/User:DavidC/30_September_2009 30]
October
MTWTFSS
      [http://2009.igem.org/User:DavidC/1_October_2009 1] [http://2009.igem.org/User:DavidC/2_October_2009 2] [http://2009.igem.org/User:DavidC/3_October_2009 3] [http://2009.igem.org/User:DavidC/4_October_2009 4]
[http://2009.igem.org/User:DavidC/5_October_2009 5] [http://2009.igem.org/User:DavidC/6_October_2009 6] [http://2009.igem.org/User:DavidC/7_October_2009 7] [http://2009.igem.org/User:DavidC/8_October_2009 8] [http://2009.igem.org/User:DavidC/9_October_2009 9] [http://2009.igem.org/User:DavidC/10_October_2009 10] [http://2009.igem.org/User:DavidC/11_October_2009 11]
[http://2009.igem.org/User:DavidC/12_October_2009 12] [http://2009.igem.org/User:DavidC/13_October_2009 13] [http://2009.igem.org/User:DavidC/14_October_2009 14] [http://2009.igem.org/User:DavidC/15_October_2009 15] [http://2009.igem.org/User:DavidC/16_October_2009 16] [http://2009.igem.org/User:DavidC/17_October_2009 17] [http://2009.igem.org/User:DavidC/18_October_2009 18]
[http://2009.igem.org/User:DavidC/19_October_2009 19] [http://2009.igem.org/User:DavidC/20_October_2009 20] [http://2009.igem.org/User:DavidC/21_October_2009 21] [http://2009.igem.org/User:DavidC/22_October_2009 22] [http://2009.igem.org/User:DavidC/23_October_2009 23] [http://2009.igem.org/User:DavidC/24_October_2009 24] [http://2009.igem.org/User:DavidC/25_October_2009 25]
[http://2009.igem.org/User:DavidC/26_October_2009 26] [http://2009.igem.org/User:DavidC/27_October_2009 27] [http://2009.igem.org/User:DavidC/28_October_2009 28] [http://2009.igem.org/User:DavidC/29_October_2009 29] [http://2009.igem.org/User:DavidC/30_October_2009 30] [http://2009.igem.org/User:DavidC/31_October_2009 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.

Retrieved from "http://2009.igem.org/User:DavidC/7_October_2009"