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
Plasmid
Terminator
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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| [http://2010.igem.org/Team:Panama/3_June_2010 3]
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/3_July_2010&action=edit 3]
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| [http://2010.igem.org/Team:Panama/29_July_2010 29]
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/4_August_2010&action=edit 4]
| [http://2010.igem.org/Team:Panama/5_August_2010 5]
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/28_August_2010&action=edit 28]
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/1_September_2010&action=edit 1]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/2_September_2010&action=edit 2]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/3_September_2010&action=edit 3]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/4_September_2010&action=edit 4]
| [http://2010.igem.org/Team:Panama/5_September_2010 5]
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| [http://2010.igem.org/Team:Panama/8_September_2010 8]
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| [http://2010.igem.org/Team:Panama/10_September_2010 10]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/11_September_2010&action=edit 11]
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| [http://2010.igem.org/Team:Panama/15_September_2010 15]
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/30_September_2010&action=edit 30]
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/1_October_2010&action=edit 1]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/2_October_2010&action=edit 2]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/3_October_2010&action=edit 3]
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/5_October_2010&action=edit 5]
| [http://2010.igem.org/Team:Panama/6_October_2010 6]
| [http://2010.igem.org/Team:Panama/7_October_2010 7]
| [http://2010.igem.org/Team:Panama/8_October_2010 8]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/9_October_2010&action=edit 9]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/10_October_2010&action=edit 10]
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| [http://2010.igem.org/Team:Panama/12_October_2010 12]
| [http://2010.igem.org/Team:Panama/13_October_2010 13]
| [http://2010.igem.org/Team:Panama/14_October_2010 14]
| [http://2010.igem.org/Team:Panama/15_October_2010 15]
| [http://2010.igem.org/Team:Panama/16_October_2010 16]
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| [http://2010.igem.org/Team:Panama/19_October_2010 19]
| [http://2010.igem.org/Team:Panama/20_October_2010 20]
| [http://2010.igem.org/Team:Panama/21_October_2010 21]
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| [http://2010.igem.org/Team:Panama/23_October_2010 23]
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| [http://2010.igem.org/Team:Panama/26_October_2010 26]
| [http://2010.igem.org/Team:Panama/27_October_2010 27]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/28_October_2010&action=edit 28]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/29_October_2010&action=edit 29]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/30_October_2010&action=edit 30]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/31_October_2010&action=edit 31]
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