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 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

Final volume 50 uL

Plasmid

Buffer 2 5 uL

BSA 0.5 uL

EcoR1 1 uL

Spe1 1 uL

DNA 20 uL

H2O 22.5 uL

Final volume 50 uL

Terminator

Buffer 2 5 uL

BSA 0.5 uL

Xba 1 uL

Pst1 1 uL

DNA 20 uL

H2O 22.5 uL

Final volume 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).

2). RhT-2b

Tm= 51.7° C

MW= 5845.9

46.6 nMoles= 9.000260

46.6 nM x 10= 466 uL H2O

Resuspending in 466 uL H2O [ ]= 100 uMolar stock

Work solution (10 uM)

VC= VC

(X)(100 uM)=(100 uL) (10 uM)

X= 10 uL of Stock

10 uL Stock + 90 uL H2O [ ]= 10 uMolar.

We tried with 3 differents volumes

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

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

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

Program

1) 94° C 5mins

2) 94° C 30seg

51° C 1mins

72° C 3mins

3) 72° C 10mins

1) H2O 6.5 uL

Primer RhT-F1b 2.5 uL

Primer RhT-2b 2.5 uL

Master mix 12.5 uL

DNA 1 uL

Final volume= 25 uL

2) H2O 9 uL

Primer RhT-2b 1.25 uL

Primer Rht-F1b 1.25 uL

Master mix 12.5 uL

DNA 1 uL

Final volume= 25 uL

3) H2O 9.5 uL

Primer F1b 1uL

Primer 2b 1mL

Master mix 12.5 uL

DNA 1 uL

Final volume= 25 uL

1% Gel Terminators (Extraction)

1. Hind III

1 uL Marker

+2 uL loading

+7 uL H2O

2. Ter A1

5uL sample

+2 uL loading

3. Ter A2

5uL sample

+2 uL loading

3. Ter K2

5uL sample

+2 uL loading