Team:Panama/10 September 2010
From 2010.igem.org
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
Plasmid
Terminator
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
PCR Reaction 2
PCR Reaction 3
1% Gel Terminators (Extraction)
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