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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 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
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