Team:Panama/10 September 2010
From 2010.igem.org
(New page: ==='''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 kan...) |
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10 uL standards + 190 uL WB. | 10 uL standards + 190 uL WB. | ||
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Measurements: | Measurements: | ||
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Terminator | Terminator | ||
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H2O 22.5 uL | H2O 22.5 uL | ||
- | + | Final volume 50 uL | |
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H2O 22.5 uL | H2O 22.5 uL | ||
- | + | Final volume 50 uL | |
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H2O 22.5 uL | H2O 22.5 uL | ||
- | + | Final volume 50 uL | |
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I. Resuspending the lyophilized primers for the solution stock (100 Plasmid u molar). | I. Resuspending the lyophilized primers for the solution stock (100 Plasmid u molar). | ||
- | 1). RhT-F1b | + | '''1). RhT-F1b''' |
Tm 49.9° C, 34.1 nMoles= 6.10D260. | Tm 49.9° C, 34.1 nMoles= 6.10D260. | ||
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(X)(100um) = (100 uL) (10 um) | (X)(100um) = (100 uL) (10 um) | ||
X = 10 uL del stock (100 uMolar). | X = 10 uL del stock (100 uMolar). | ||
+ | |||
+ | '''2). RhT-2b''' | ||
+ | |||
+ | Tm= 51.7° C | ||
+ | |||
+ | MW= 5845.9 | ||
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+ | 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 |
Revision as of 22:51, 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
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