용액속에 들어가서 해당 농도가 되어야 하는지 해당 농도의 시약을 사용해서
buffer를 만들었는지는 2가지 모두 가능하기 때문에 원문을 봐야 알수 있습니다.
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레벨1
파닥파닥
(대학생)
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19.12.02 10:53
논문 페이지가 많아 업로드가 안되는거 같아서 본문 복붙하였습니다.
Materials TE buffer (APPENDIX 2) 10% sodium dodecyl sulfate (SDS) 20 mg/ml proteinase K (stored in small single-use aliquots at −20°C) 5 M NaCl CTAB/NaCl solution 24:1 chloroform/isoamyl alcohol 25:24:1 phenol/chloroform/isoamyl alcohol (UNIT 2.1) Isopropanol 70% ethanol
1. Inoculate a 5-ml liquid culture with the bacterial strain of interest. Grow in conditions appropriate for that strain (i.e., appropriate medium, drug selection, temperature) until the culture is saturated. This may take several hours to several days, depending on the growth rate.
2. Spin 1.5 ml of the culture in a microcentrifuge for 2 min, or until a compact pellet forms. Discard the supernatant.
3. Resuspend pellet in 567 µl TE buffer by repeated pipetting. Add 30 µl of 10% SDS and 3 µl of 20 mg/ml proteinase K to give a final concentration of 100 µg/ml proteinase K in 0.5% SDS. Mix thoroughly and incubate 1 hr at 37°C. The solution should become viscous as the detergent lyses the bacterial cell walls. There should be no need to predigest the bacterial cell wall with lysozyme.
4. Add 100 µl of 5 M NaCl and mix thoroughly. This step is very important since a CTAB–nucleic acid precipitate will form if salt concentration drops below about 0.5 M at room temperature (Murray and Thompson, 1980). The aim here is to remove cell wall debris, denatured protein, and polysaccharides complexed to CTAB, while retaining the nucleic acids in solution.
출처: Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J.G. Seidman, John A. Smith, Kevin Struhl (eds.) Current Protocols in Molecular Biology Copyright © 2003 John Wiley & Sons, Inc. All rights reserved.
제가 보았을때는 각각의 농도로 희석된 시약을 따로따로 첨가하는거 같은데
저 시약들을 한꺼번에 합쳐 lysis buffer를 제조시에는 buffer 총량에 맞게 농도가 희석되게끔 시약을 넣어 제조하면 되는건가요?
10% SDS를 30ul 사용했고 TE buffer의 1mM EDTA는 일반적으로 용액에 final
1mM EDTA 농도로 사용할겁니다.
또한 TE 10 : 0.1농도로 사용하는 경우도 있으며 이때는 EDTA 0.1mM이 최종농도
입니다.