Difference between revisions of "Team:Nagahama/Design"
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=== Transformation by heat shock=== | === Transformation by heat shock=== | ||
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== isoprenoid production == | == isoprenoid production == | ||
Revision as of 02:49, 14 September 2015
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)
↓Remove supernatant
↓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
Proof of concept(POC)
isoprenoid production
Because of its high hydrophobicity and low volatility, decane was chosen to extract and solubilize farnesolFOH from the culture broth. The decane was overlaidy in the two-phase culture media, but it did not affect the cell growth., and FOH farnesol could be well solubilized in the decane phase with negligible volatile loss. We adopt used 1 mL of decane to overlayid to the 5 mL of culture broth. Two-phase The culture of E. coli JM109 (BBa_K165025) was carried out in 2YT medium containing 1% glycerol at 29°C for 48 h. The decane phase of the two-phase culture was collected to analyze the FOH content by GC-MS.