Team:Panama/10 September 2010

From 2010.igem.org

(Difference between revisions)
(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...)
(→September 10)
Line 21: Line 21:
10 uL standards + 190 uL WB.
10 uL standards + 190 uL WB.
 +
Measurements:
Measurements:
 +
Terminator
Terminator
Line 56: Line 58:
H2O          22.5 uL         
H2O          22.5 uL         
-
VF            50  uL         
+
Final volume  50  uL         
Line 73: Line 75:
H2O          22.5 uL       
H2O          22.5 uL       
-
VF            50  uL       
+
Final volume  50  uL       
Line 90: Line 92:
H2O          22.5 uL         
H2O          22.5 uL         
-
VF            50  uL         
+
Final volume  50  uL         
Line 109: Line 111:
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.
Line 127: Line 129:
(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
 +
 +
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

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