User:DavidC/1 October 2009

From 2009.igem.org

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=== Thursday the 1st ===
 +
 +
==== PCR ====
 +
 +
==== D protein of Lambda phage ====
 +
 +
Primers : <br>
 +
Forward : Pref_ATG_Fw <br>
 +
Reverse : Suffixe_Rv <br><br>
 +
 +
DNA : <br>
 +
Purified and amplified D protein from Lambda phage. <br><br>
 +
 +
Cycles: <br>
 +
94°C 2minutes; <br>
 +
(94°C 1 minute; <br>
 +
59°C 1 minutes; <br>
 +
72°C 1 minute(s)) X 35; <br>
 +
72°C 4 minutes; <br>
 +
 +
==== Adénovirus 5 penton base ====
 +
 +
Primers: <br>
 +
Forward : Pref_ATG_Fw <br>
 +
Reverse : Suffixe_Rv <br><br>
 +
 +
DNA : <br>
 +
Purified and amplified penton base from the adenovirus 5. <br><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>
 +
 +
==== Ligation of D protein and adenovirus 5 penton base ====
 +
 +
Restriction digest of samples by SpeI and XbaI. <br><br>
 +
 +
Primers: <br>
 +
Forward : Pref_ATG_Fw <br>
 +
Reverse : Suffixe_Rv <br><br>
 +
 +
DNA: <br>
 +
1. D protein restriction digests by SpeI and Adenovirus 5 penton base digests by XbaI. <br>
 +
2. D protein and Adenovirus 5 penton base digest by BalI. <br><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>
 +
 +
==== DNA purification ====
 +
 +
No purification, We obtain negative results. <br>
 +
 +
==== DNA extraction ====
 +
 +
Miniprep (promega): <br>
 +
 +
Samples: <br>
 +
BBa_C0012 + BBa_B0014 ; <br>
 +
BBa_C0012. <br>
 +
 +
==== Restriction digest ====
 +
 +
BBa_C0012 + BBa_B0014 digest by EcoRI and and XbaI (3302bp): <br>
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) =4µL <br>
 +
H20 = 4µL <br>
 +
EcoR1 (TAKARA) = 1µL <br>
 +
Xba1 (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== Ligation between COS and pSB1A2 backbone ====
 +
 +
COS insert (purified PCR product) digests by XbaI and PstI (250bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) =4µL <br>
 +
H20 = 4µL <br>
 +
Xba1 (TAKARA) = 1µL <br>
 +
Pst1 (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
pSB1A2 digests by XbaI and PstI (2079bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) =4µL <br>
 +
H20 = 4µL <br>
 +
Xba1 (TAKARA) = 1µL <br>
 +
Pst1 (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 COS and pSB1A2: ====
 +
 +
First report : <br>
 +
COS = 4µL <br>
 +
pSB1A2 = 1µL <br>
 +
Solution A = 20µL <br>
 +
Solution B = 5µL <br>
 +
 +
Second report : <br>
 +
COS = 2µL <br>
 +
pSB1A2 = 0,5µL <br>
 +
Solution A = 10µL <br>
 +
Solution B = 2,5µL <br>
 +
 +
Third report : <br>
 +
COS = 6µL <br>
 +
pSB1A2 = 1µL <br>
 +
Solution A = 24µL <br>
 +
Solution B = 6µL <br>
 +
 +
1 hour of incubation at room temperature <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>

Latest revision as of 03:56, 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

Thursday the 1st

PCR

D protein of Lambda phage

Primers :
Forward : Pref_ATG_Fw
Reverse : Suffixe_Rv

DNA :
Purified and amplified D protein from Lambda phage.

Cycles:
94°C 2minutes;
(94°C 1 minute;
59°C 1 minutes;
72°C 1 minute(s)) X 35;
72°C 4 minutes;

Adénovirus 5 penton base

Primers:
Forward : Pref_ATG_Fw
Reverse : Suffixe_Rv

DNA :
Purified and amplified penton base from the adenovirus 5.

Cycles:
94°C 2minutes;
(94°C 1 minute;
59°C 1 minutes;
72°C 2 minute(s)) X 35;
72°C 5 minutes;

Ligation of D protein and adenovirus 5 penton base

Restriction digest of samples by SpeI and XbaI.

Primers:
Forward : Pref_ATG_Fw
Reverse : Suffixe_Rv

DNA:
1. D protein restriction digests by SpeI and Adenovirus 5 penton base digests by XbaI.
2. D protein and Adenovirus 5 penton base digest by BalI.

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.

DNA purification

No purification, We obtain negative results.

DNA extraction

Miniprep (promega):

Samples:
BBa_C0012 + BBa_B0014 ;
BBa_C0012.

Restriction digest

BBa_C0012 + BBa_B0014 digest by EcoRI and and XbaI (3302bp):
DNA (miniprep) = 30µL
Buffer M (TAKARA) =4µL
H20 = 4µL
EcoR1 (TAKARA) = 1µL
Xba1 (TAKARA) = 1µL
1 hour of incubation at 37°C.

Ligation between COS and pSB1A2 backbone

COS insert (purified PCR product) digests by XbaI and PstI (250bp):

DNA (miniprep) = 30µL
Buffer M (TAKARA) =4µL
H20 = 4µL
Xba1 (TAKARA) = 1µL
Pst1 (TAKARA) = 1µL
1 hour of incubation at 37°C.

pSB1A2 digests by XbaI and PstI (2079bp):

DNA (miniprep) = 30µL
Buffer M (TAKARA) =4µL
H20 = 4µL
Xba1 (TAKARA) = 1µL
Pst1 (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 COS and pSB1A2:

First report :
COS = 4µL
pSB1A2 = 1µL
Solution A = 20µL
Solution B = 5µL

Second report :
COS = 2µL
pSB1A2 = 0,5µL
Solution A = 10µL
Solution B = 2,5µL

Third report :
COS = 6µL
pSB1A2 = 1µL
Solution A = 24µL
Solution B = 6µL

1 hour of incubation at room temperature

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).

Retrieved from "http://2009.igem.org/User:DavidC/1_October_2009"