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Team:Panama/10 September 2010
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Ter K2 ---> Terminator grown in medium with kanamycin. | Ter K2 ---> Terminator grown in medium with kanamycin. | ||
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DNA quantification with Qubit fluorometer (invitrogen). | DNA quantification with Qubit fluorometer (invitrogen). | ||
| - | Samples | + | '''Samples''' |
| - | + | 1uL sample + 199uL WB. | |
| - | + | '''Standards''' | |
| - | + | 10uL standards + 190uL WB. | |
| - | Measurements: | + | '''Measurements:''' |
Terminator | Terminator | ||
| - | 24. | + | 24.9ug/mL T1 (A1). |
| + | 19.4ug/mL T2 (A2). | ||
| - | + | 32.5ug/mL T3 (K1). | |
| + | 28.7ug/mL T4 (K2). | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
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| + | '''Agarose gel 1%.''' | ||
Digest, Terminator & Reporter reactions. | Digest, Terminator & Reporter reactions. | ||
| - | '''Reporter | + | '''Reporter''' |
| - | Buffer 2 | + | Buffer 2 5uL |
| - | BSA 0. | + | BSA 0.5uL |
| - | EcoR1 | + | EcoR1 1uL |
| - | Spe1 | + | Spe1 1uL |
| - | DNA | + | DNA 20uL |
| - | H2O 22. | + | H2O 22.5uL |
| - | Final volume | + | Final volume 50uL |
'''Plasmid''' | '''Plasmid''' | ||
| - | Buffer 2 | + | Buffer 2 5uL |
| - | BSA 0. | + | BSA 0.5uL |
| - | EcoR1 | + | EcoR1 1uL |
| - | Spe1 | + | Spe1 1uL |
| - | DNA | + | DNA 20uL |
| - | H2O 22. | + | H2O 22.5uL |
| - | Final volume | + | Final volume 50uL |
'''Terminator''' | '''Terminator''' | ||
| - | Buffer 2 | + | Buffer 2 5uL |
| - | + | ||
| - | + | ||
| - | + | BSA 0.5uL | |
| - | + | Xba 1uL | |
| - | + | Pst1 1uL | |
| - | + | DNA 20uL | |
| - | + | H2O 22.5uL | |
| + | Final volume 50uL | ||
| - | |||
| - | It takes 20 minutes at 80° C to deactivate enzymes. | + | This was placed 3 hours at 37° C on the thermocycler. It takes 20 minutes at 80° C to deactivate enzymes. |
| - | PCR | + | PCR gDNA. Pseudomona (Rh1AB) |
| - | II Test. | + | II Test. Pair of primers: |
Primers: | Primers: | ||
| Line 251: | Line 244: | ||
| - | [[Image:DSC02620.JPG| | + | [[Image:DSC02620.JPG|250px|thumb|left|alt text]] |
Revision as of 13:05, 26 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
1uL sample + 199uL WB.
Standards
10uL standards + 190uL WB.
Measurements:
Terminator
24.9ug/mL T1 (A1).
19.4ug/mL T2 (A2).
32.5ug/mL T3 (K1).
28.7ug/mL T4 (K2).
Agarose gel 1%.
Digest, Terminator & Reporter reactions.
Reporter
Buffer 2 5uL
BSA 0.5uL
EcoR1 1uL
Spe1 1uL
DNA 20uL
H2O 22.5uL
Final volume 50uL
Plasmid
Buffer 2 5uL
BSA 0.5uL
EcoR1 1uL
Spe1 1uL
DNA 20uL
H2O 22.5uL
Final volume 50uL
Terminator
Buffer 2 5uL
BSA 0.5uL
Xba 1uL
Pst1 1uL
DNA 20uL
H2O 22.5uL
Final volume 50uL
This was placed 3 hours at 37° C on the thermocycler. It takes 20 minutes at 80° C to deactivate enzymes.
PCR gDNA. Pseudomona (Rh1AB)
II Test. Pair 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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