IGEM:PennState/2007/Dnaprep: Difference between revisions
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(New page: Miniprep of Bacterial Genomic DNA #Grow bacterial strain to saturation #Spin 1.5 mL for 2 min in microcentrifuge #Resuspend in 567 ul TE buffere, 30ul of 20 mg/ml proteinase K. Mix and in...) |
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#Spin 1.5 mL for 2 min in microcentrifuge | #Spin 1.5 mL for 2 min in microcentrifuge | ||
#Resuspend in 567 ul TE buffere, 30ul of 20 mg/ml proteinase K. Mix and incubate 1 hr at 37C | #Resuspend in 567 ul TE buffere, 30ul of 20 mg/ml proteinase K. Mix and incubate 1 hr at 37C | ||
#*http://www.neb.com/nebecomm/products/productP8102.asp | |||
#Add 100ul of 5M NaCl. Mix thoroughly | #Add 100ul of 5M NaCl. Mix thoroughly | ||
#Add 80 ul of CTAB/NaCl solution. Mix. Incubate 10 min at 65C. | #Add 80 ul of CTAB/NaCl solution. Mix. Incubate 10 min at 65C. | ||
#*CTAB/NaCl solution (0.7M NaCl, 10% CTAB): Dissolve 4.1 g NaCl in 80 ml water. Slowly add 10 g CTAB. Stir with heat to dissolve. Bring volume to 100 ml. | |||
#Extract with an equal volume of chloroform/isoamyl alcohol. Spin 5 min in microcentrifuge. | #Extract with an equal volume of chloroform/isoamyl alcohol. Spin 5 min in microcentrifuge. | ||
#Transfer aqueous phase to a fresh tube. Precipitate DNA with .6 vol isopropanol. Wash precipitate with 70% ethanol. Remove supernatant and briefly dry pellet in lyophilizer. | #Transfer aqueous phase to a fresh tube. Precipitate DNA with .6 vol isopropanol. Wash precipitate with 70% ethanol. Remove supernatant and briefly dry pellet in lyophilizer. | ||
#Resuspend pellet in 100ul TE buffer. | #Resuspend pellet in 100ul TE buffer. | ||
Revision as of 12:47, 20 June 2007
Miniprep of Bacterial Genomic DNA
- Grow bacterial strain to saturation
- Spin 1.5 mL for 2 min in microcentrifuge
- Resuspend in 567 ul TE buffere, 30ul of 20 mg/ml proteinase K. Mix and incubate 1 hr at 37C
- Add 100ul of 5M NaCl. Mix thoroughly
- Add 80 ul of CTAB/NaCl solution. Mix. Incubate 10 min at 65C.
- CTAB/NaCl solution (0.7M NaCl, 10% CTAB): Dissolve 4.1 g NaCl in 80 ml water. Slowly add 10 g CTAB. Stir with heat to dissolve. Bring volume to 100 ml.
- Extract with an equal volume of chloroform/isoamyl alcohol. Spin 5 min in microcentrifuge.
- Transfer aqueous phase to a fresh tube. Precipitate DNA with .6 vol isopropanol. Wash precipitate with 70% ethanol. Remove supernatant and briefly dry pellet in lyophilizer.
- Resuspend pellet in 100ul TE buffer.
