Team:Nagahama/colombia
Contents
Uniandes_Colombia
Participation of low budget
We helped Uniandes Colombia. Low budget which began it from last year. The purpose of the low budget is other iGEM team creat new protocole which is more cheaper, easy and quickly than original. and iGEM teams that do not have enough funds to pay reagent products they can use them and othere igem team. We participated it. We send The protocol of DNA extraction with alkaline extraction. Kit extraction by DNA extraction, takes most cost. The DNA extracted by alkaline extraction is a simple protocol with much lower cost than the Kit Extraction.
Protocol for low Budget
Comparison of the protocol and the price
The DNA extraction with Kit extraction (NucleoSpin® Plasmid EasyPure TAKARA)
↓1.5ml tube
↓＋Culture 1.5ml
↓Centrifuge 12000 g 30sec
↓Collect the supernatant and cast aside
↓＋A1 buffer 150μl
↓＋A2 buffer 250μl
↓Room temperature 2min
↓＋A3 buffer 350μl
↓Gently mix
↓Centrifuge 12000 g 3min
↓Column into a collection tube and decant the supernatant
↓Centrifuge 1000~2000 g 30sec
↓＋450μl AQ Buffer
↓Centrifuge 12000 g 1min
↓37℃ to dry 10~15min
↓New 1.5ml tube
↓Set column
↓＋50μl AE buffer
↓Room temperature 1min
↓Centrifuge 12000 g 1min
↓4 ℃ preserve Refrigerator
total price 420￥≒3.5＄
The DNA extraction with alkaline extraction
↓1.5 ml tube
↓＋1.5 ml culture
↓Centrifuge 13000 rpm 1min 4℃
↓Collect the supernatant and cast aside
↓＋Soln 1: 200μl mix by voltex
↓＋soln 2: 200μl
↓Gently mix by inverting the tube
↓＋soln 3: 200μl
↓Gently mix
↓On ice 5min
↓Centrifuge 13000 rpm 10 min 4℃
↓Collect the supernatant and add to the new tube
↓＋phenol、chloroform、isoamylalcohol 500μl
↓Mix by voltex
↓Centrifuge 13000 rpm 5 min 4℃
↓Take only the only water layer
↓add to the new tube
↓＋100 % Etoh 400 μl
↓Mix by vortex
↓Centrifuge 13000 rpm 20 min 4 ℃
↓Throw away the supernatant to alcohol waste bin
↓ ＋70 % Etoh 500 μl
↓Gently mix
↓Throw away the supernatant to alcohol waste bin
↓65 ℃ to dry
↓＋TE buffer 50 μl
↓＋RNase A (0.5 μg/μl)
↓37 ℃ 20 min
↓Voltex and Tabletop small centrifuge
↓4 ℃ preserve Refrigerator
Reagent
TE buffer 1ml ( dH2O 985μl , 1M Tris-HCl (pH8.0) 10μl , 200mM EDTA 5μl)
Soln 1: 1 ml (dH2O 900μl , 1M Tris-HCl (pH8.0) 50μl , 200mM EDTA 50μl)
Soln 2: 1 ml (dH2O 876μl , 5M NaOH 24μl , 10%SDS 100μl )
Soln 3: 1 ml (dH2O 290μl , 5M potassium acetate , 600μl , acetic acid 110μl)
RNase A: (0.5 μg/μl) 1μl
Reagent price
1M Tris-HCl 60μl : 3￥ ≒ 0.03＄
200mM EDTA 55μl : 0.396￥ ≒ 0.003＄
5M NaOH 24μl : 0.028￥≒0.0002＄
10%SDS 100μl : 1.8￥≒0.0149＄
5M potassium acetate 600μl : 1.65￥≒0.0136＄
acetic acid 110μl : 0.18￥≒0.0015＄
RNase A (0.5 μg/μl) 1μl : 130￥≒1.1＄
Tube ×3 : 6.5￥≒0.054＄
Total price 144￥≒1.22＄