Team:Imperial College/Growth

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Cell Count v Optical Density Curve Calibration

Aim

To produce a calibration curve to aid in the normalising of fluorescence values to allow proper characterisation of Promoters and RBSs for B.subitlis. This protocol must give results that are as accurate as possible over a considerable range of Optical Densities.

Equipment

Spectrophotometer
Cuvettes
P20, P200 and P1000 Gilsons

Reagents

20ml of LB in a 250ml flask
B. subtilis transformed with antibiotic selectable marker
LB agar plates with antibiotic

Protocol

Inoculate 20ml of LB broth with B. subtilis containing a selectable marker, we used B. subtilis transformed with spectinomycin. Incubate overnight at 37^oC.
Collect culture and measure the O.D.₆₀₀.
With the O.D.₆₀₀ calculate how much of the overnight culture is required to give an O.D.₆₀₀ of 0.5, 1, 1.5, 2, 2.5, 3 in 2ml using the following calculation:
- Vol of culture (ml)=(O.D.₆₀₀ wanted / O.D.₆₀₀ of Overnight culture) * 2ml
Carry out the dilutions required using LB media to make the volumes up to 2ml.
Remove 1ml of each dilution and then measure the O.D.₆₀₀ and discard.
Now carry out a series of 10 fold dilutions by removing 100ul of the dilution and adding into to 900ul of LB (this gives 10^-1 dilution) carry this on until a suitable dilution is reached. We used the following dilutions:
- O.D₆₀₀ of 0.5 = x10^-6 and x10^-7
- O.D₆₀₀ of 1 = x10^-6 and x10^-7
- O.D₆₀₀ of 1.5 = x10^-6 and x10^-7
- O.D₆₀₀ of 2 = x10^-6 and x10^-7
- O.D₆₀₀ of 2.5 = x10^-6 and x10^-7
- O.D₆₀₀ of 3 = x10^-7 and x10^-8
Plate 100ul of the appropriate dilutions onto an LB agar plate containing the antibiotic selectable marker.
Grow the plates at 37°C overnight
The following day count the number of colonies on the plates and multiply up by the dilution to obtain the colony forming units at each time point. Remember when plating the cells only 100ul of 1000ul was used to plate and so you need to multiply by a further 10.

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 {{Imperial/StartPage2}}__NOTOC__
-=Cell Count v Optical Density Curve Calibration=
-===Aim===
+{{Imperial/Box1|Cell Count v Optical Density Curve Calibration|
+======Aim======
 To produce a calibration curve to aid in the normalising of fluorescence values to allow proper characterisation of Promoters and RBSs for ''B.subitlis''. This protocol must give results that are as accurate as possible over a considerable range of Optical Densities.
+======Equipment======
-===Equipment===
 *Spectrophotometer
 *Cuvettes
 *P20, P200 and P1000 Gilsons
+======Reagents======
-===Reagents===
 *20ml of LB in a 250ml flask
-*''B.subtilis'' transformed with antibiotic selectable marker
+*''B. subtilis'' transformed with antibiotic selectable marker
-*LB agar plates with anitbiotic
+*LB agar plates with antibiotic
+======Protocol======
-===Protocol===
+#Inoculate 20ml of LB broth with ''B. subtilis'' containing a selectable marker, we used ''B. subtilis'' transformed with spectinomycin. Incubate overnight at 37<sup>o</sup>C.
-*Innoculate 20ml of LB broth with ''B.subtilis'' containing a selectable marker, we used ''B.subtilis'' transformed with spectinomycin. Incubate overnight at 37<sup>o</sup>C.
+#Collect culture and measure the O.D.<sub>600</sub>.
-*Collect culture and measure the O.D.<sub>600</sub>.
+#With the O.D.<sub>600</sub> calculate how much of the overnight culture is required to give an O.D.<sub>600</sub> of 0.5, 1, 1.5, 2, 2.5, 3 in 2ml using the following calculation:
-*With the O.D.<sub>600</sub> calculate how much of the overnight culture is required to give an O.D.<sub>600</sub> of 0.5, 1, 1.5, 2, 2.5, 3 in 2ml using the following calculation:
+#*Vol of culture (ml){{Equals}}(O.D.<sub>600</sub> wanted / O.D.<sub>600</sub> of Overnight culture) * 2ml
-**Vol of culture (ml)=(O.D.<sub>600</sub> wanted / O.D.<sub>600</sub> of Overnight culture) * 2ml
+#Carry out the dilutions required using LB media to make the volumes up to 2ml.
-*Carry out the dilutions required using LB media to make the volumes upto 2ml.
+#Remove 1ml of each dilution and then measure the O.D.<sub>600</sub> and discard.
-*Remove 1ml of each dilution and then measure the O.D.<sub>600</sub> and discard.
+#Now carry out a series of 10 fold dilutions by removing 100ul of the dilution and adding into to 900ul of LB (this gives 10<sup>-1</sup> dilution) carry this on until a suitable dilution is reached. We used the following dilutions:
-*Now carry out a series of 10 fold dilutions by removing 100ul of the dilution and adding into to 900ul of LB (this gives 10<sup>-1</sup> dilution) carry this on until a suitable dilution is reached. We used the following dilutions:
+#*O.D<sub>600</sub> of 0.5 {{Equals}} x10<sup>-6</sup> and x10<sup>-7</sup>
-**O.D<sub>600</sub> of 0.5 = x10<sup>-6</sup> and x10<sup>-7</sup>
+#*O.D<sub>600</sub> of 1 {{Equals}} x10<sup>-6</sup> and x10<sup>-7</sup>
-**O.D<sub>600</sub> of 1 = x10<sup>-6</sup> and x10<sup>-7</sup>
+#*O.D<sub>600</sub> of 1.5 {{Equals}} x10<sup>-6</sup> and x10<sup>-7</sup>
-**O.D<sub>600</sub> of 1.5 = x10<sup>-6</sup> and x10<sup>-7</sup>
+#*O.D<sub>600</sub> of 2 {{Equals}} x10<sup>-6</sup> and x10<sup>-7</sup>
-**O.D<sub>600</sub> of 2 = x10<sup>-6</sup> and x10<sup>-7</sup>
+#*O.D<sub>600</sub> of 2.5 {{Equals}} x10<sup>-6</sup> and x10<sup>-7</sup>
-**O.D<sub>600</sub> of 2.5 = x10<sup>-6</sup> and x10<sup>-7</sup>
+#*O.D<sub>600</sub> of 3 {{Equals}} x10<sup>-7</sup> and x10<sup>-8</sup>
-**O.D<sub>600</sub> of 3 = x10<sup>-7</sup> and x10<sup>-8</sup>
+#Plate 100ul of the appropriate dilutions onto an LB agar plate containing the antibiotic selectable marker.
-*Plate 100ul of the appropriate dilutions onto an LB agar plate containing the antibiotic selectable marker.
+#Grow the plates at 37°C overnight
-*Grow the plates at 37°C overnight
+#The following day count the number of colonies on the plates and multiply up by the dilution to obtain the colony forming units at each time point. Remember when plating the cells only 100ul of 1000ul was used to plate and so you need to multiply by a further 10.
-*The following day count the number of colonies on the plates and multiply up by the dilution to obtain the colony forming units at each time point. Remember when plating the cells only 100ul of 1000ul was used to plate and so you need to multiply by a further 10.
+|}}
 {{Imperial/EndPage|Protocols|}}