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Revision as of 03:36, 7 September 2016

Week 10: 8/29 - 9/4

Week 10: 8/29 - 9/4



8/29

AmilCP BioBrick Digests

Content Experimental ul Control ul
H2O 46.5 47.0
Cutsmart 5.0 5.0
XbaI 0.5 X
DNA 2.0 2.0

PCR of Toggle 1

Contents ul
Phire 10
H2O 8
5 uM F Primer 1.5
5 uM R Primer 1.5
100pg/ul Construct 1.0

  • Ran other trials with 2.5, 3.5, 4.5 and 6.5 ul of Forward and Reverse Primers
  • Water ul was adjusted to compensate: 6, 4, 2 and 0 ul respectively
Phase Temperature (°C) Time (sec)
Initial Denaturation 98 30
Denaturation 98 5
Annealing 68 5
Extension 72 45
Final Extension 72 60

PCR of Toggle 2

Contents ul
Phire 10
H2O 8
5 uM F Primer 1.5
5 uM R Primer 1.5
100pg/ul Construct 1.0

  • Ran other trials with 2.5, 3.5, 4.5 and 6.5 ul of Forward and Reverse Primers
  • Water ul was adjusted to compensate: 6, 4, 2 and 0 ul respectively
Phase Temperature (°C) Time (sec)
Initial Denaturation 98 30
Denaturation 98 5
Annealing 68 5
Extension 72 45
Final Extension 72 60

Digests of Toggle parts

Toggle 1 Digest
Content Experimental ul Control ul
H2O 24 25
Cutsmart 5.0 5.0
SpeI 1.0 X
DNA 25.0 25.0
Toggle 1 Digest
Content Experimental ul Control ul
H2O 24 25
Cutsmart 5.0 5.0
XbaI 1.0 X
DNA 25.0 25.0

Linearized pSB1C7 Digest


Content Experimental ul Control ul
H2O 16.5 18.5
Cutsmart 2.5 2.5
SpeI-HF 1.0 X
XbaI 1.0 X
DNA 4.0 4.0


Ligation of Toggle parts

Content ul
10X T4 Buffer 2.0
Toggle 1 5.0
Toggle 2 10.0
H2O 2.0
Ligase 1.0


8/30

Ligate Toggle 2 into Plasmid

    Content ul
    Ligase Buffer 2
    Toggle pieces 10 (500 ng/ul)
    Plasmid 2
    H2O 5
    Ligase 1

Phosphatase of linearized pSBK3

    Amount ul
    Digestion of plasmid 10
    Buffer 2
    Phosphatase 1
    H2O 7

Ligate into Plasmid treated with Phosphatase

    Amount ul
    Plasmid 5
    Toggle pieces 10 (500 ng/uL)
    Buffer 2
    H2O 2
    Ligase 1


8/31

Digest of IDT CR-1 SBU and Vector

CR-1 SBU
Content Experimental ul Control ul
H2O 18 20
Cutsmart 5.0 5.0
EcoRI-HF 1.0 X
XhoI 1.0 X
DNA 25.0 25.0
pEP352GAP
Content Experimental ul Control ul
H2O 7.0 8.0
Cutsmart 1.0 1.0
EcoRI-HF 0.5 X
XhoI 0.5 X
DNA 1.0 1.0

Incubation

  • Incubated overnight


9/1

Deactivation of Digest with Phosphatase



PCR of Toggle #1

  • Same setup as 8/30

Ligation of CR-1 into pEP352GAP



9/2

PCR of Toggle 1

Contents ul
Phire 10
H2O 8.0
5 uM F Primer 0.5
5 uM R Primer 0.5
100pg/ul DNA 1.0
  • Also ran trials with 1.5, 2.5, 3.5 and 4.5 ul of both forward and reverse primers
  • Adjusted H2O accordingly
PCR Settings: 30 cycles
Phase Temperature (°C) Time (sec)
Initial Denaturation 98 30
Denaturation 98 10
Annealing 67 5
Extension 72 25
Final Extension 72 60

Another PCR of Toggle 1

Contents ul
Phire 10
H2O 7.74
10 uM F Primer 0.63
10 uM R Primer 0.63
100pg/ul DNA 1.0
  • Ran trials with 1.25 and 1.88 ul of forward and reverse primers
  • H2O for those trials were 6.5 and 5.24 ul respectively
PCR Settings: 35 cycles
Phase Temperature (°C) Time (sec)
Initial Denaturation 98 30
Denaturation 98 10
Annealing 68.5 5
Extension 72 20
Final Extension 72 60

PCR of Toggle 2

Contents ul
Phire 10
H2O 7.74
10 uM F Primer 0.63
10 uM R Primer 0.63
100pg/ul DNA 1.0
  • Ran trials with 1.25 and 1.88 ul of forward and reverse primers
  • H2O for those trials were 6.5 and 5.24 ul respectively
PCR Settings: 35 cycles
Phase Temperature (°C) Time (sec)
Initial Denaturation 98 30
Denaturation 98 10
Annealing 68.5 5
Extension 72 35
Final Extension 72 60


9/3



9/4