Team:Wash U/Protocol
From 2009.igem.org
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'''Solution C''' | '''Solution C''' | ||
Make up to 4 Litres | Make up to 4 Litres | ||
- | + | *Nitrilotriacetic acid 40g | |
- | + | *Magnesium Chloride MgCl2 96g | |
- | + | *Calcium Chloride CaCl2 13.36g | |
- | + | *EDTA 0.5g | |
- | + | *Zinc Chloride (poison) ZnCl2 1.044g | |
- | + | *Ferrous Chloride (poison) FeCl2 1.0g | |
- | + | *Manganous Chloride MnCl2 0.36g | |
- | + | *Ammonium molybdate (NH4)6Mo7O244H2O 0.037g | |
- | + | *Cupric Chloride (poison) CuCl2 0.031g | |
- | + | *Cobaltous nitrate (poison) Co(NO3)2 0.0496g | |
- | + | *Boric acid (orthoboric acid) 0.0228g | |
Do not autoclave, just freeze at -20°C in 400ml aliquots. | Do not autoclave, just freeze at -20°C in 400ml aliquots. | ||
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Casamino acids (CAA): | Casamino acids (CAA): | ||
To make 1 Litre | To make 1 Litre | ||
- | + | *Casein Hydrosylate acid 50g | |
Makes up 5% solution to be aliquotted into 200ml. | Makes up 5% solution to be aliquotted into 200ml. | ||
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1X M22: | 1X M22: | ||
To make up 2 Litres | To make up 2 Litres | ||
- | + | *10X stock M22 200ml | |
- | + | *CAA 40ml | |
- | + | *Water 1760ml | |
Batches - 1.5 Litres in 2 Litre flasks | Batches - 1.5 Litres in 2 Litre flasks | ||
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prepare a 10,000 times stock solution of vitamins as follows. | prepare a 10,000 times stock solution of vitamins as follows. | ||
- | nicotinic acid 1g | + | *nicotinic acid 1g |
- | Thiamine 0.5g | + | *Thiamine 0.5g |
- | pABA(p-Aminobenzoic acid) 0.1g | + | *pABA(p-Aminobenzoic acid) 0.1g |
- | Biotin (d-Biotin) 0.01g | + | *Biotin (d-Biotin) 0.01g |
- | Milli-Q water 100ml | + | *Milli-Q water 100ml |
Aliquot into 20mls after filter sterilisation. | Aliquot into 20mls after filter sterilisation. | ||
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- | Conjugation | + | Conjugation: Triparental Mating |
- | + | *Plasmid (pRK404 derived) in E. coli GC5 (or other F- strain) | |
- | + | *Conjugation helper strain: E. coli with pRK2013 | |
- | + | *Destination strain: R. sphaeroides DBCΩ(LH2 Knockout with streptomycin resistance) | |
- | + | *Expect to see colonies in 4-6 days | |
- | + | ||
Conjugation Procedure | Conjugation Procedure | ||
- | 1) Start cultures | + | 1) Start cultures |
- | + | *Start 15ml 2 day culture R. sphaeroides DBCΩ with strep 10ug/ml in M22 | |
- | + | *Start 15 ml overnight culture E. coli GC5 with plasmid to transfer in LB with strep 5ug/ml tet 5ug/ml | |
- | + | *Start 15ml overnight culture E. coli with pRK2013 in LB with kan 50ug/ml | |
2) Spin down cultures for 5 minutes | 2) Spin down cultures for 5 minutes | ||
3) Remove supernatant from cultures and resuspend in 1ml antibiotic-free liquid media | 3) Remove supernatant from cultures and resuspend in 1ml antibiotic-free liquid media | ||
- | + | *Use LB for E. coli | |
- | + | *Use M22 for R. sphaeroides DBCΩ | |
4) Combine equal volumes of resuspended cells (~30ul each) | 4) Combine equal volumes of resuspended cells (~30ul each) | ||
5) Pipette mixture of cells onto well dried M22 plate | 5) Pipette mixture of cells onto well dried M22 plate |
Revision as of 20:46, 18 October 2009