User:Howard Boland/Notebook/Art from Synthetic Biology/2011/02/02

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BBa_I13504+BBa_katE
The following steps were performed towards a new composited part

Check overnight growth (BBa_I13504)
Growth worked fine - all three tubes grew.

Take Glycerol Stock (BBa_I13504)
For each tubes make glycerol stock
 * 1) 700µl Bacteria Broth
 * 2) 300µl 50% Glycerol

Miniprep samples(BBa_I13504)
Purify plasmids using QIAGen Miniprep kit
 * 1) Eluted in 35µl Water

Digestion
4 Digestions where setup
 * 1) BBa_katE - the pcr product was digested with EcoRI and SpeI (Upstream)
 * 2) BBa_I13504 was digested with XbaI and PstI

Gel
The digestion was run on a 2% Gel. Check against BBa_I13504 sample
 * 1) Lane 1: 100bp ladder
 * 2) Lane 2: Blank
 * 3) Lane 3: 50µl digested sample BBa_katE- E/S (~505bp)
 * 4) Lane 4: Blank
 * 5) Lane 5: 50µl digested sample BBa_katE- E/S (~505bp)
 * 6) Lane 6: Blank
 * 7) Lane 7: 50µl digested sample 1 BBa_I13504- X/P (875bp)
 * 8) Lane 8: 50µl digested sample 2 BBa_I13504- X/P (875bp)



5x M9 Salt, 200ml
Minimal media M9 salt, at pH7
 * 1) 1.2g di-Sodium hydrogen orthophosphate (Na2HPO4, 6g/l)
 * 2) 0.6g Potassium dihydrogen phosphate (KH2PO4,  3g/l)
 * 3) 0.1g NaCl (0.5g/l)
 * 4) 0.2g Ammonium chloride (NH4Cl, 1g/l)

Autoclave at 121ºC for 15 minutes

M9 Media, 200ml

 * 1) 150ml H2O, Distilled (Cooled to <50ºC)
 * 2) 40ml 5x M9 Salt
 * 3) 200µl 1M MgSO4 (1ml/l)
 * 4) 0.2g Casamino acids (1g/l)
 * 5) 20µl 1M CaCl2 (0.1ml/l)
 * 6) Adjust volume to 200ml using sterile H2O

Carbon Sources

 * 1) 1% Glucose Or 0.5% Glycerol

5 bottles, 10ml 50% Glycerol

 * 1) Add 5ml Glycerol
 * 2) Add 5ml water

Autoclave 20min in liquid cycle

Effector

 * 1) 3.6mM IPTG
 * 2) 5.3mM cAMP

0.1M CaCl2, 500ml
Used to prepare competent cells.

CaClx6H2O has a molecular weight of FW 219.08.


 * 1) To make 1M in one litre of water add 219.08g powder and increase volume by adding water to 1litre.
 * 2) To make 0.1M in 200ml of water add 0.1*(219.08g)/(1000ml/500ml) = 0.1*219.08g / 2 = 10.95g powder and increase volume by adding water up to 500ml.

Autoclave at 121ºC for 15minutes

0.1M MgCl2, 500ml
Used to prepare competent cells.

MgCl2x6H2O has a molecular weight of FW 203.3.


 * 1) To make 1M in one litre of water add 203.3g powder and increase volume by adding water to 1litre.
 * 2) To make 0.1M in 200ml of water add 0.1*(203.3g)/(1000ml/500ml) = 0.1*203.3g / 2 = 10.16g powder and increase volume by adding water up to 500ml.

Autoclave at 121ºC for 15minutes

2x 500ml water
Autoclave at 121ºC for 15minutes

Locate Potassium L-lactate
I am currently missing this substrate to grow my nanomagentic bacteria.

Test FSM with Pyruvic as Carbon Source
Since I have not ben able to locate lactate I am testing pryuvic acid as a carbon source.

To 200ml FSM I added 0.47g powder
 * pryuvic acids = 88.06 g/mol
 * 27mM in 200ml => (0.027)*88.06g/(1000ml/200ml)=0.47g


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