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From 2010.igem.org

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Revision as of 22:12, 14 October 2010

September 10

Ter A1 ---> Terminator grown in medium with ampicillin.

Ter A2 ---> Terminator grown in medium with ampicillin.

Ter K1 ---> Terminator grown in medium with kanamycin.

Ter K2 ---> Terminator grown in medium with kanamycin.

DNA quantification with Qubit fluorometer (invitrogen).

Samples

1 uL sample + 199 uL WB.

Standars

10 uL standards + 190 uL WB. Measurements: Terminator

24.9 ug/mL T1 (A1).

19.4 ug/mL T2 (A2).

32.5 ug/mL T3 (K1).

28.7 ug/mL T4 (K2).

Agarose gel 1%.

Digest, Terminator & Reporter reactions.

Reporter

Buffer 2 5 uL

BSA 0.5 uL

EcoR1 1 uL

Spe1 1 uL

DNA 20 uL

H2O 22.5 uL

VF 50 uL

Plasmid

Buffer 2 5 uL

BSA 0.5 uL

EcoR1 1 uL

Spe1 1 uL

DNA 20 uL

H2O 22.5 uL

VF 50 uL

Terminator

Buffer 2 5 uL

BSA 0.5 uL

Xba 1 uL

Pst1 1 uL

DNA 20 uL

H2O 22.5 uL

VF 50 uL

This was placed 3 hours at 37° C on the thermocycler.

It takes 20 minutes at 80° C to deactivate enzymes.

PCR DNAg. Pseudomona (Rh1AB)

II Test. Par of primers:

Primers:

RhT-F1b5´ GTT TGC CTG TTC GAA AAT T 3´

RhT-2b 5´ CGA TAC GGC AAA ATC ATG G 3´

I. Resuspending the lyophilized primers for the solution stock (100 Plasmid u molar).

1). RhT-F1b

Tm 49.9° C, 34.1 nMoles= 6.10D260.

MW 5808.8

34.1 nMoles X 10= 341 uL H2O

Resuspending with 341 uL H2O [ ]= 100 uMolar stock.

Work solution (10 uMolar)

10 uL (Stock 100 uMolar) + 90 uL H2O [ ]= 10 uMolar

VC = VC (X)(100um) = (100 uL) (10 um) X = 10 uL del stock (100 uMolar).