Team:Newcastle/Labwork/10 August 2009

=Formal Lab Session - 10th August 2009= = Overview = 
 * Stochastic Switch Team - midi-prepped pGFP-rrnB plasmid which had been grown up in E. coli cells
 * Sporulation Tuning/Chassis Team - had a second attempt at making buffer solution (with MgCl2) needed for Method A and also made up some LB + Kan + Em plates
 * Metal Sensor Team - prepared for test Bacillus subtilis transformations by carrying out 'Eveing before transformations' step in protocol

Stochastic Switch Team
Today we prepared pGFP-rrnB plasmids using midiprep. We followed GenElute's midiprep protocol.

We had two E.coli cultures inoculated with pGFP-rrnB left overnight. Although the samples were of the same plasmid, we treated them individually as one culture seemed to express the GFP more than the other. We used 100ml of each sample aliquoted into two 50ml falcon tubes then carried out the midi prep procedure from the protocol on the 4 tubes, combining our DNA samples at the end. In the DNA concentration step we centrifuged the tubes at 16000rpm for 30 minutes before adding ethanol to rinse the DNAs. We then centriguged the tubes again for 10 minutes at 16000rpm.

We evaporated the tubes for 5 minutes at the end before resuspending each tube in 50ul of distilled water. The tubes from the same cultures were then combined and the DNA concentrations for the two samples were measured using the spectrometer.

Results
We got good DNA concentrations for both samples. The results are below:

Sample 1
The concentration of DNA: 829.6 ng/uL The ratio of DNA concentration to protein concentration(260/280): 1.88 The ratio of protein concentration to RNA concentration(260/230): 2.33



Sample 2
The concentration of DNA: 601.1 ng/uL The ratio of DNA concentration to protein concentration(260/280): 1.90 The ratio of protein concentration to RNA concentration(260/230): 2.11



Summary
Today, we made our second attempt at making the buffer solution for the recovery of the spores sent to us by Anne Moir from Sheffield University. We also decided to prepare the plates for plating out the sleB and cwlJ spores.



As Dr. Phil Aldrige's lab did not have stock of Em which is required, Em was borrowed from Prof. Colin Harwood's lab. On the 4th of August, the buffer solution was prepared, however it was prepared wrongly, and therefore remade again today. For the buffer solution, as seen in the protocol for Method A, MgCl is required and a stock solution has already been made for it. However, upon inspection, we realised that the MgCl stock solution has been contaminated, probably due to the fact that it was made several years ago, and thus we made a new 0.05M stock solution of MgCl. A stock solution of 1M KCl was already kindly made by someone else from Dr. Phil Aldrige's lab, and thus we made up the last required solution, 0.1M stock solution of Potassium Phosphate. The stock solutions we prepared were then sent for autoclaving. We decided to pour 500ml of LB with Em and Kan. We were earlier taught how to pour 1 litre of media correctly, and were confident in doing so. However, we failed to realise that at half the amount during practice, we should not let the LB + agar media cool to too low a temperature. Therefore, when we mixed the cooled DI Water with the LB + agar media, the solution solidified. However, we persevered and successfully poured the plates the following day.

Buffer Solution for Spores Recovery
*0.05M MgCl2 stock solution *0.1M Potassium Phosphate stock solution
 * Items to be Prepared


 * 0.05M MgCl2 Stock Solution

MW * Desired Volume (L) * Desired Molarity (M)

203.3 * 0.25L * 0.05M = 25.413g

Therefore, 25.413g of MgCl2 is mixed with 0.25L of DI water to make 0.25L of 0.05M MgCl2.


 * 0.1M Potassium Phosphate Stock Solution

MW * Desired Volume (L) * Desired Molarity (M)

174.18 * 0.25L * 0.1M = 4.355g

Therefore, 4.355g of Potassium Phosphate is mixed with 0.25L of DI water to make 0.25L of 0.1M Potassium Phosphate.

The stock solutions are to be sent for autoclaving before use.

After the stock solutions have been autoclaved, we prepared the buffer solution for the recovery of the spores. As not much of the buffer solution is required, we decided to make 100ml of it.

The final concentration of each component as seen in the protocol for Method A should be:
 * 0.01M Potassium Phosphate
 * 0.05M KCl
 * 0.001MgCl2

Therefore,

Emr Stock Solution for Addition to Agar Plates

 * Emr Stock Solution
 * 50mg of Em in 50ml of DI water

Required amount of Emr = 1ug/ml Stock solution of Emr = 1mg/ml
 * Amount of Emr Stock Solution to be Added

Therefore, 1mg / 1ug = 1000 The stock solution needs to be diluted 1000 times to obtain the correct concentration of 1ug/ml.

500ml of LB + Kan + Emr is to be poured, therefore: 500ml / 1000 = 0.5ml 0.5ml of Emr is to be added to 500ml of LB + agarose solution.

Summary
Preparation for test transformation in Bacillus subtilis 168 with GFP-rrnB plasmid DNA This afternoon we prepared 40 ml MM competence media and equally distributed it into four 15ml falcon tubes. To three of the tubes (one tube for each of the three teams), Bacillus subtilis 168 was added and to the fourth tube, nothing was added - this would serve as contamination control. They were then left in the shaking incubator at 37ºC overnight for the transformation process tomorrow.

Protocol
For the preparation of the 40ml MM competence media (10ml in each of 4 falcon tubes), please refer to the solutions list. For the steps regarding preparations of overnight cultures, please refer to the 'Evening before transformations day' section. The only changes to the protocol were: 4 falcon tubes were used instead of just 2 - three tubes contained Bacillus subtilis innoculated MM competence media (for the three sub-teams in our project) and the fourth tube contained MM competence media alone (contamination control)

Results
3 x 15ml falcon tubes containing 10ml MM competence media along with Bacillus subtilis cells and 1 x 15ml falcon tube containing just 10ml MM competence media. All four tubes placed in shaking incubator for overnight growth.