Using biobrick DNA recovered from registry on 23/7/2008
Do PCR with I0500, gel electrophoresis and recover DNA from gel using Gel DNA recovery kit
Biobrick BBa_1006 of about 250bp used as reference
DNA run on 0.8% agarose gel with SyBr Green
PCR protocol with 34 cycles:
5μL DNA
1μL of each primer VR and VF2
3μL SDW
10μL Finnzyme Master Mix
Total volume of 20μL
Protocol for preparation of SyBr Green 0.8% Agarose Gel:
0.8g of Ultrapure agarose powder
Add 100ml of TAE
Microwave to dissolve agarose powder
Add 100μL SyBr Green when bottle is cool enough to hold
Gel should be about 6mm deep
Run gel with 80V
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Lane 3 - Hyperladder I<br>
Lane 4 - I746:101<br>
Lane 5 - I746:001<br>
Lane 7 - I0500 promoter PCR product<br>
Lane 8 - I006 control<br>
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Result:
Bright band of about 1.2kb found on gel corresponding to I0500
Preparation of M9 Medium (Iron Free)
M9 salt and glucose solution is reduced in iron content using Chelex-100 resins, which are removed by filtration.
Millipore filter used to pass the prepared M9 medium (glucose added) to sterilise the medium at the end.
Sterilised deionised water should be used in the preparation of iron citrate stock.
Preparation of M9 Medium
5X M9 salts contain:
64 g Na2HPO4.7H2O
15 g KH2PO4
2.5 g NaCl
5 g NH4Cl
For the M9 medium,
Component Concentrations:
1x M9 salts
2mM MgSO4
0.1mM CaCl2
In SDW
For 500ml of media:
250ml 2xM9 salts
10ml 0.1M MgSO4 (MgSO4•7H2O used so 0.493g added to 20ml H2O)
100μl 0.5M CaCl2 (0.055g anhydrous CaCl2 into 1000μl H2O)
234.9ml sterile deionized H2O
5ml 40% glucose (4g glucose in 6ml H2O)
Protocol:
To make up a 5x M9 concentrate we need to add 56.4g in 1L H2O. Thus for 2xM9 we need to dissolve 11.28g M9 into 500ml H2O
Disslove glucose into the M9 salt solution
Pass M9 salts and glucose solution through 50ml Chelex-100 columns to reduce iron content of reagents before use
Residual chelex 100 removed by filtration
Dissolve other components into solution
Preparation of 200ml iron citrate stock (iron:citrate ratio = 1:100)
Reagents needed:
1. Tri-sodium citrate
CAS No: 68-04-2
Hazard: Irritation to skin, eyes and respiratory tract
400mM needed
Dissolve 23.528g of Na3C6H5O7 sodium citrate into 200ml distilled deionized water
2. Ferrous sulfate FeSO4•7H2O
CAS No. 7720-78-7
Hazard: Irritation to skin, eyes and respiratory tract
4mM needed
0.222g of FeSO4•7H2O dissolved in 200ml of aqueous sodium citrate solution to give 4mM FeSO4
3. Aqueous sodium hydroxide
250ml of 0.5M NaOH needed to adjust stock to pH7
Dissolve 5g of NaOH into 250ml of distilled deionised water
Hazard: corrosive
Ferrous sulfate should be added when needed in the experiment to prevent oxidation of Fe(II) to insoluble Fe(III) precipitates.
Anaerobic condition needed when incubating E.coli to prevent such oxidation reaction which affects soluble iron ion concentration in solution and the iron uptake by bacteria.