User:DavidC/2 October 2009

From 2009.igem.org

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=== Friday the 2nd ===
 +
 +
==== Restriction digest ====
 +
 +
==== Ligation between BBa_B0030 and BBa_R0010 ====
 +
 +
Restriction digest of R0010 by SpeI and PstI (2279bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer H (TAKARA) =4µL <br>
 +
H20 = 4µL <br>F
 +
Spe1 (TAKARA) = 1µL <br>
 +
Pst1 (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br><br>
 +
 +
Restriction digest of BBa_B0030 by PstI and XbaI (15bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) =4µL <br>
 +
H20 = 4µL <br>
 +
Pst1 (TAKARA) = 1µL <br>
 +
Xba1 (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== Ligation between BBa_B0014 and BBa_P1003 + BBa_B0014 ====
 +
 +
Restriction digest of BBa_B0014 by EcoRI and SpeI (95bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) =4µL <br>
 +
H20 = 4µL <br>
 +
EcoR1 (TAKARA) = 1µL <br>
 +
Spe1 (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br><br>
 +
 +
Restriction digst of BBa_P1003 + BBa_B0014 by EcoRI and XbaI (3770bp): <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><br>
 +
 +
==== Ligation between COS and pSB1A2 ====
 +
 +
Restriction digest of COS by XbaI and SpeI (250bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) =4µL <br>
 +
H20 = 4µL <br>
 +
Xba1 (TAKARA) = 1µL <br>
 +
Spe1 (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br><br>
 +
 +
Restriction digest of pSB1A2 by SpeI and XbaI (2079bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) =4µL <br>
 +
H20 = 4µL <br>
 +
Spe1 (TAKARA) = 1µL <br>
 +
Xba1 (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br><br>
 +
 +
==== Ligation of the D protein (PrtD) with the adenovirus 5 penton base (ADV5pb) ====
 +
 +
Restriction digest of PrtD by SpeI (385bp): <br>
 +
 +
DNA (PCR) = 5µL <br>
 +
Buffer M (TAKARA) =2µL <br>
 +
H20 = 12µL <br>
 +
Spe1 (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br><br>
 +
 +
Restriction digest of ADV5pb by XbaI (1715bp): <br>
 +
 +
DNA (PCR) = 5µL <br>
 +
Buffer M (TAKARA) =2µL <br>
 +
BSA (TAKARA) = 2µL <br>
 +
H20 = 10µL <br>
 +
Xba1 (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br><br>
 +
 +
==== DNA electrophoresis ====
 +
 +
85 Volt, 15 minutes. <br>
 +
105 Volt, 40 minutes. <br>
 +
Ladder fermentas 1 Kb. <br>
 +
 +
 +
Samples: BBa_R0010, BBa_B0030, BBaB0014, BBa_P1003, adenovirus penton base with BioBrick prefix and suffix (ADV5pb BBa), D protein with BioBrick prefix and suffix (D prt BBa)
 +
<br>
 +
 +
[[image:F0210.png|center]]
 +
<br>
 +
 +
Interpretation:
 +
 +
We obtain all the fragments but we always have a problem of mistmacth for ADV5 pb primers, and the B0030 fragment is impossible to see into the photo, in fact there is a few DNA, and the fragment is short around 15bp.
 +
 +
==== DNA purification ====
 +
 +
Kit Qiagen “gel extraction kit”, final volume = 50µL. <br>
 +
 +
==== Ligation ====
 +
 +
Ligation between COS and pSB1A2 plasmid backbone (TAKARA, DNA ligation kit) : <br>
 +
 +
First report : <br>
 +
COS = 3,5µL <br>
 +
pSB1A2 = 0,5µL <br>
 +
Solution A = 16µL <br>
 +
Solution B = 4µL <br>
 +
 +
Second report: <br>
 +
COS = 6µL <br>
 +
pSB1A2 = 1µL <br>
 +
Solution A = 28µL <br>
 +
Solution B = 7µL <br>
 +
 +
1 hours of incubation at room temperature <br><br>
 +
 +
Ligation between the D protein and ADV5pb (NEB enzyme): <br>
 +
 +
First report : <br>
 +
PrtD = 2,5µL <br>
 +
ADV5pb = 5,5µL <br>
 +
NEB T4 ligase buffer= 1µL <br>
 +
NEB T4 ligase = 1µL <br><br>
 +
 +
Second report: <br>
 +
 +
PrtD = 3µL <br>ADV5pb = 5µL <br>
 +
NEB T4 ligase buffer= 1µL <br>
 +
NEB T4 ligase = 1µL <br><br>
 +
 +
Third report: <br>
 +
PrtD = 4µL <br>
 +
ADV5pb = 4µL <br>
 +
NEB T4 ligase buffer= 1µL <br>
 +
NEB T4 ligase = 1µL <br><br>
 +
 +
Fourth report: <br>
 +
PrtD = 2µL <br>
 +
ADV5pb = 2µL <br>
 +
NEB T4 ligase buffer= 1µL <br>
 +
NEB T4 ligase = 1µL <br>
 +
 +
1 hour at room temperature <br><br>
 +
 +
Ligation between BBa_B0030 and BBa_R0010 (TAKARA, DNA ligation kit) : <br>
 +
 +
First report : <br>
 +
Plasmid (R0010) = 0,5µL <br>
 +
Insert (B0030) = 5µL <br>
 +
Solution A = 22µL <br>
 +
Solution B = 5,5µL <br><br>
 +
 +
Second report: <br>
 +
Plasmid (R0010) = 0,5µL <br>
 +
Insert (B0030) = 6µL <br>
 +
Solution A = 26µL <br>
 +
Solution B = 6,5µL <br>
 +
 +
1 hour of incubation at room temperature. <br><br>
 +
 +
Ligation between BBa_B0014 and BBa_P1003 + BBa_B0014 (TAKARA, DNA ligation kit) : <br>
 +
 +
First report : <br>
 +
Plasmid (P1003 + B0014) = 0,5µL <br>
 +
Insert (B0014) = 5µL <br>
 +
Solution A = 22µL <br>
 +
Solution B = 5,5µL <br>
 +
 +
Second report : <br>
 +
Plasmid (P1003 + B0014) = 0,5µL <br>
 +
Insert (B0014) = 6µL <br>
 +
Solution A = 26µL <br>
 +
Solution B = 6,5µL <br>
 +
 +
1 hour of incubation at room temperature. <br><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 16:05, 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

Friday the 2nd

Restriction digest

Ligation between BBa_B0030 and BBa_R0010

Restriction digest of R0010 by SpeI and PstI (2279bp):

DNA (miniprep) = 30µL
Buffer H (TAKARA) =4µL
H20 = 4µL
F Spe1 (TAKARA) = 1µL
Pst1 (TAKARA) = 1µL
1 hour of incubation at 37°C.

Restriction digest of BBa_B0030 by PstI and XbaI (15bp):

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

Ligation between BBa_B0014 and BBa_P1003 + BBa_B0014

Restriction digest of BBa_B0014 by EcoRI and SpeI (95bp):

DNA (miniprep) = 30µL
Buffer M (TAKARA) =4µL
H20 = 4µL
EcoR1 (TAKARA) = 1µL
Spe1 (TAKARA) = 1µL
1 hour of incubation at 37°C.

Restriction digst of BBa_P1003 + BBa_B0014 by EcoRI and XbaI (3770bp):

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

Restriction digest of COS by XbaI and SpeI (250bp):

DNA (miniprep) = 30µL
Buffer M (TAKARA) =4µL
H20 = 4µL
Xba1 (TAKARA) = 1µL
Spe1 (TAKARA) = 1µL
1 hour of incubation at 37°C.

Restriction digest of pSB1A2 by SpeI and XbaI (2079bp):

DNA (miniprep) = 30µL
Buffer M (TAKARA) =4µL
H20 = 4µL
Spe1 (TAKARA) = 1µL
Xba1 (TAKARA) = 1µL
1 hour of incubation at 37°C.

Ligation of the D protein (PrtD) with the adenovirus 5 penton base (ADV5pb)

Restriction digest of PrtD by SpeI (385bp):

DNA (PCR) = 5µL
Buffer M (TAKARA) =2µL
H20 = 12µL
Spe1 (TAKARA) = 1µL
1 hour of incubation at 37°C.

Restriction digest of ADV5pb by XbaI (1715bp):

DNA (PCR) = 5µL
Buffer M (TAKARA) =2µL
BSA (TAKARA) = 2µL
H20 = 10µL
Xba1 (TAKARA) = 1µL
1 hour of incubation at 37°C.

DNA electrophoresis

85 Volt, 15 minutes.
105 Volt, 40 minutes.
Ladder fermentas 1 Kb.


Samples: BBa_R0010, BBa_B0030, BBaB0014, BBa_P1003, adenovirus penton base with BioBrick prefix and suffix (ADV5pb BBa), D protein with BioBrick prefix and suffix (D prt BBa)

F0210.png


Interpretation:

We obtain all the fragments but we always have a problem of mistmacth for ADV5 pb primers, and the B0030 fragment is impossible to see into the photo, in fact there is a few DNA, and the fragment is short around 15bp.

DNA purification

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

Ligation

Ligation between COS and pSB1A2 plasmid backbone (TAKARA, DNA ligation kit) :

First report :
COS = 3,5µL
pSB1A2 = 0,5µL
Solution A = 16µL
Solution B = 4µL

Second report:
COS = 6µL
pSB1A2 = 1µL
Solution A = 28µL
Solution B = 7µL

1 hours of incubation at room temperature

Ligation between the D protein and ADV5pb (NEB enzyme):

First report :
PrtD = 2,5µL
ADV5pb = 5,5µL
NEB T4 ligase buffer= 1µL
NEB T4 ligase = 1µL

Second report:

PrtD = 3µL
ADV5pb = 5µL
NEB T4 ligase buffer= 1µL
NEB T4 ligase = 1µL

Third report:
PrtD = 4µL
ADV5pb = 4µL
NEB T4 ligase buffer= 1µL
NEB T4 ligase = 1µL

Fourth report:
PrtD = 2µL
ADV5pb = 2µL
NEB T4 ligase buffer= 1µL
NEB T4 ligase = 1µL

1 hour at room temperature

Ligation between BBa_B0030 and BBa_R0010 (TAKARA, DNA ligation kit) :

First report :
Plasmid (R0010) = 0,5µL
Insert (B0030) = 5µL
Solution A = 22µL
Solution B = 5,5µL

Second report:
Plasmid (R0010) = 0,5µL
Insert (B0030) = 6µL
Solution A = 26µL
Solution B = 6,5µL

1 hour of incubation at room temperature.

Ligation between BBa_B0014 and BBa_P1003 + BBa_B0014 (TAKARA, DNA ligation kit) :

First report :
Plasmid (P1003 + B0014) = 0,5µL
Insert (B0014) = 5µL
Solution A = 22µL
Solution B = 5,5µL

Second report :
Plasmid (P1003 + B0014) = 0,5µL
Insert (B0014) = 6µL
Solution A = 26µL
Solution B = 6,5µL

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/2_October_2009"