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

Reactive..........1x [ul]
 * For plasmid 18 II

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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