Team:Newcastle/Labwork/7 August 2009

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=Formal Lab Session - 7th August 2009=
==<u>Stochastic Switch Team</u>==
==<u>Stochastic Switch Team</u>==

Revision as of 15:53, 6 October 2009

Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG

Formal Lab Session - 7th August 2009

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 ww will use gfp-rrnb integration vector. It uses spectinamycin as the antibiotic,

We solved 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.

Preparation

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.




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