From 2009.igem.org
| August
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| M | T | W | T | F | S | S
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| [http://2009.igem.org/User:DavidC/1_August_2009 1]
| [http://2009.igem.org/User:DavidC/2_August_2009 2]
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| [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]
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| [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]
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| [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]
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| [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]
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| [http://2009.igem.org/User:DavidC/31_August_2009 31]
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| September
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| M | T | W | T | F | S | S
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| [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]
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| [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]
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| [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]
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| [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]
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| [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]
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| October
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| M | T | W | T | F | S | S
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| [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]
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| [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]
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| [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]
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| [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]
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| [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]
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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):
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)
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).
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