Team:Newcastle/Labwork/7 August 2009

=Formal Lab Session - 7th August 2009= = Overview =  MAIN EMPHASIS WAS ON MODELLING TODAY
 * Stochastic Switch Team - prepared some spectinomycin solution
 * Sporulation Tuning/Chassis Team - correctly calculated and made up chemicals needed for Method A of recovering spores

Stochastic Switch Team
Today, we identified the strains and prepared the spectinamycin solution.

We will use the E.coli strain numbered as 2386.

B. subtilis 168 strain is already in the fridge. To test our biopbricks we will use pGFP-rrnb integration vector. It uses spectinamycin as the antibiotic,

We dissolved 65mg of the spectinamycin in 13 ml of water, purified the solution and placed it into the fridge.

Prepared volume of the spectinamycin solution: 13 ml Concentration: 5mg/ml

Sporulation Tuning/Chassis Team
Today, we prepared Lysozyme stock solution in preparation for the recovery of our cwlD spores kindly sent to us by Anne Moir from Sheffield University.

Referring to Method A of the protocol, it states that there should be 200ug per ml of buffer solution present, and 10mM of L-alanine to initiate germination.

The following paragraphs shows the calculations of stock solutions made, and how much solution to add to obtain final concentrations desired.

Lysozyme
We need: 200ug Lysozyme in 1000ul DI Water 0.2mg Lysozyme in 1ml DI Water

We made 0.5g of Lysozyme in 10ml of DI Water which we filter sterilised, where: 0.5g Lysozyme in 10ml DI Water 500mg in 10ml DI Water = 50mg Lysozyme in 1ml DI Water 50 000ug in 1000ul DI Water = 50ug Lysozyme in 1ul DI Water

Therefore, the amount of Lysozyme stock solution to add to a ml of buffer solution is: 50ug (Stock Solution) / 2ug (Desired Molarity) = 25 In order to obtain a solution with a final concentration of 2ug/ul, the stock solution needs to be diluted 25 times. Desired volume = 1ml 1000ul / 25 = 40ul

4ul 40ul of Lysozyme stock solution is needed per ml of buffer solution.