Team:Nagahama/Design
Contents
Protocols
Our Lab's Protocols
Medium
LB medium (100 mL liquid)
1.Measure 1g Tripton
2.Measure 0.5g Yeast Extract
3.Measure 1g Nacl
4.Add 100mL H2O
5.autoclave(121℃ 20min)
2×YT medium (100mL liquid)
1.Measure 1.6g Tripton
2.Measure 1g Yeast Extract
3.Measure 0.5g Nacl
4.Add 100mL H2O
5.autoclave(121℃ 20min)
DNA work
Agarose gel(100mL)
Method of Making 0.7% Agarose gel
1.Measure 0.7g Agarose
2.Add 100mL TAE buffer
3.Heat(till agarose melted)*We used a microwave oven.
4.Pur agarose into a gel maker
5.Set a comb
6.Wait till agarose curdles
7.Pull an comb
Genome DNA extraction
↓Cultivate E. coli DH5α using LB medium 2ml×2 tubes O/N
↓Centrifuge culture 1.5ml (13,000rpm 4℃ 1min)
↓Remove the culture
↓+TNE buffer 1.0ml (10mM Tris pH 8.0, 10 mM NaCl, 10 mM EDTA)
↓vortex
↓Centrifuge cell suspension (13,000rpm 4℃ 2min)
↓Remove supernatant
↓+TNE buffer including 1% Triton X-100 270µl
↓Suspend cell gently
↓+5mg/ml Lysozyme solution 30µl (0.15g Lyzozyme + 30µl sterile water)
↓Reaction 37℃ 30min
↓+ 20mg/ml Proteinase K (fin.con: 1mg/ml) TaKaRa Code:9033
↓Reaction 65℃ 2h
↓+Phenol chloroform 300µl ...(1)
↓Mix the solution gently ...(2)
↓Centrifuge (13,000rpm 4℃ 8min) ...(3)
↓Transfer only water layer to new 1.5ml tube ...(4)
↓Repeat (1)~(4) 2 times
↓+3M Sodium acetate 30µl
↓Mix gently
↓+99.5% EtOH 750µl
↓Mix gently
↓Centrifuge (13,000rpm 4℃ 10min)
↓Remove supernatant
↓+70% EtOH 500µl
↓Centrifuge (13,000rpm 4℃ 1min)
↓Dry up the pellet covering with aluminum foil at room temperature 30min
↓+TE buffer 50µl
↓Suspend DNA gently
Plasmids extraction
PCR
Transformation by heat shock
isoprenoid production
geraniol tolerant assay
GC
GC-MS
Experiments & Protocols
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