Team:Panama/10 September 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 concentration measurements:

DNA quantification with Qubit fluorometer (Invitrogen).

Plasmid Terminator Concentrations:

24.9ug/mL T1 (A1).

19.4ug/mL T2 (A2).

32.5ug/mL T3 (K1).

28.7ug/mL T4 (K2).

Agarose gel 1%.

Digestion Reactions of Plasmid,Reporter & Terminator.

Reporter

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Plasmid

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Terminator

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This was placed for 3 hours at 37°C on the thermocycler. It takes 20 minutes at 80°C to deactivate enzymes.

Rh1AB(Pseudomona) PCR amplification: Second Test

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 341uL H2O [ ]= 100 uMolar stock.

Work solution (10 uMolar)

2). RhT-2b

Tm= 51.7° C

MW= 5845.9

46.6 nMoles= 9.000260

46.6 nM x 10= 466uL H2O

Resuspending in 466uL H2O [ ]= 100 uMolar stock

Work solution (10 uM)

We tried with 3 differents volumes

1) 2.5uL [1 um] Final volume= 25uL

2) 1.25uL [0.5 um] Final volume= 25uL

3) 1uL [0.4 um] Final volume= 25uL

Thermocycler Program

1) 94° C 5 mins.

2) 94° C 30 segs.

51° C 1 min.

72° C 3 min.

3) 72° C 10 mins.

PCR Reaction 1

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PCR Reaction 2

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PCR Reaction 3

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1% Gel Terminators (Extraction)

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