User:Jorge Arturo Zepeda/Notebook/iGem LCG-UNAM team 2010/2010/07/01

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Thursday 1st July 2010

I performed 3 digestions of the 3 plasmids for posterior ligations as follows:

  • For plasmid 18

Reactive..........1x [ul]
DNA................25
BSA.................1
Buffer2............4
H2O................6
EcoRI-HF........2
PstI.................2
Total.............40

  • For plasmid 18 II

Reactive..........1x [ul]
DNA................25
BSA.................1
Buffer2............4
H2O................6
EcoRI-HF........2
PstI.................2
Total.............40

  • For cI inverter PCR

Reactive........1x [ul]
DNA..............10
BSA...............1
Buffer2..........3
H2O..............13
XbaI..............1.5
PstI...............1.5
Total.............30

I also did a hot start PCR (with rtth polymerase) to introduce the trpLp promoter into a medium copy number plasmid with a reverse primer containing the promoter primed at the prefix and a forward one primed at the suffix.

  • 1º Reaction

Reactive.........................1x...............7.x
H2O...............................8ul.............56ul
Buffer 3.3x.....................6ul.............42ul
Mg(OAc)2.......................3ul.............21ul
dNTP’s...........................5ul.............35ul
Oligo up-suffix..............3ul.............21ul
Oligo down-preffx.........3ul.............21ul
DNA...............................2ul..............-
Total............................30ul.............210ul

  • 2º Reaction

Reactive...........1x (ul)........7x (ul)
H2O.................10.5...........73.5
Buffer 3.3x.......9................63
Rtth .................0.5............3.5
Total................20.............140



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