Team:Imperial College London/Wetlab/Protocols/Abs

=Absorbance calibration=

Aim

 * To produce a calibration curve to aid in the normalising of absorbance values. The relation of absorbance reading to number of cells varies with different cell strains. We are therefore doing one for Top-10.

Assay
Cultures of the E.coli with the relevant vector are grown to various cell densities. A sample of these cultures are taken and a dilution plate is carried out to work out approximate colony forming units per ml of culture. This data set is then combined with absorbance readings to create a graph relating the number of colony forming cells per ml to their absorbance measurements.

This curve then allows us to convert absorbance of a known volume of culture to colony forming units within the culture sample.

Equipment

 * Multi-plate reader
 * 15ml falcon tubes
 * 96 well plates

Reagents
(for M9 minimal media preparation, refer to Secondary Carbon Sources)
 * M9 Minimal Media with 0.2% Casamino acids, 0.5% Glucose and Strep
 * LB media (with Strep)
 * LB agar plates (with Strep) x20

Things needed

 * LB + Strep

Day1: Inoculation of cells
1)Using a loop, pick out a single colony of Top-10 cells from a Top-10 Strep plate (in cold room). Innoculate the  cells on LB broth (with Strep)  and grow them overnight at 37 °C with spinning.

Day 2
1)	Before noon, check that the starter culture of Top-10 in LB has grown (ie. Turned cloudy). Take out and transfer to cold room (Because appeared not to grow in M9 directly, otherwise skip straight to step 2 below)

2)	At 3pm, dilute cells 1:50 (cells: media) into 10ml (5ml if starter media, see above) of supplemented M9 media. Grow them overnight at 37 °C

Things needed

 * Multi-well plate reader
 * 96 well plate
 * 20 LB agar plates with strep

Monitering of OD
1. When the culture has reached a high OD (>2.0), 200ul of cell culture is transferred into a 96 well plate and the absorbance at 600nm is measured using a multi-plate reader (or spectrophotometer). 2. If the OD of the culture is not very high, there is also the option of spinning the cells down using a centrifuge. The cells can then be resuspended in a smaller volume of medium. (optional step, perhaps better if step 1 reaches the high OD naturally) 3. 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) * 400ul Carry out the dilutions required using M9 media+0.5% glucose. Note 1: There will be a tendency for OD readings to be higher than expected ie. when OD 3.0 is halved, it will probably give an OD of around 2.2 rather than 1.5. Note 2: The most important region of OD to get data points should be where we expect to start collecting fluorescence readings and other data eg 1.2 Note 3: For higher values (>1 u of OD) linearity of the relationship between OD and Cell number becomes an issue. Dilute down and compensate on paper by multiplying by the dilution factor 4. Remove 200ul of each dilution and pipette into a 96 well plate. Measure the O.D.600 using a multiplate reader and discard. 5. Now carry out a series of 10 fold dilutions by removing 10ul of the dilution and adding into to 1ml of M9 (this gives 10-2 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

6. Plate 100ul of the appropriate dilutions onto an LB agar plate containing strep (including 1 replicate) Leave on bench to dry Note: These dilutions were taken from imperial 08. However, when I plated them, the agar plates were too dense. Could be technique or perhaps more dilutions are required/ less volume to be plated. 7. Grow the plates at 37°C overnight in incubator.

Colony counting
1. The following day count the number of colonies on the plates. Take them out of the incubator earlier so that they won't multiply and beome uncountable.

2.Multiply up the colonies counted 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.

Data manipulation
The absorbance is related to the number of colony forming units in a graph. The equation of the straight line was used to convert all absorbance measurements to CFU/well.