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Team:British Columbia/Protocols/DNAPrepBacteria
From 2012.igem.org
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#Grow bacteria in 50 ml LB with antibiotic to late log phase or stationary phase, harvest cells at 40C, and centrifuge at 4000 rpm for 10 minutes. | #Grow bacteria in 50 ml LB with antibiotic to late log phase or stationary phase, harvest cells at 40C, and centrifuge at 4000 rpm for 10 minutes. | ||
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#Resuspend cell pellet in 5 ml of Solution I (50mM glucose, 25 mM Tris-HCl, pH8.0, 10 mM EDTA).Add lysozyme to a final concentration of 5 mg/ml, shake at 370C for 30 minutes. | #Resuspend cell pellet in 5 ml of Solution I (50mM glucose, 25 mM Tris-HCl, pH8.0, 10 mM EDTA).Add lysozyme to a final concentration of 5 mg/ml, shake at 370C for 30 minutes. | ||
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#Add 600 ul of 10% SDS and 300 ul of proteinase K (stock concentration 20 mg/ml) to the above mixture and let stand at 550C for 1 hour. | #Add 600 ul of 10% SDS and 300 ul of proteinase K (stock concentration 20 mg/ml) to the above mixture and let stand at 550C for 1 hour. | ||
| - | + | #Extract DNA by adding 5 ml of TE-saturated phenol-chloroform mix well and spin at 4000 rpm for 10 minutes or longer to get a good separation. Transfer the aqueous to a clean tube and repeat the same extraction step two more times. | |
| - | #Extract DNA by adding 5 ml of TE-saturated phenol-chloroform mix well and spin at 4000 rpm for 10 minutes or longer to get a good separation. Transfer the aqueous to a clean tube and repeat the same extraction step two more times | + | #Transfer the aqueous phase to a clean tube and add 250 ul of 5M NaCl and 10 ml of 100% ethanol to precipitate DNA. Spin at 40C for 10 min with a speed of 2000 rpm-5000 rpm. |
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| - | #Transfer the aqueous phase to a clean tube and add 250 ul of 5M NaCl and 10 ml of 100% ethanol to precipitate DNA. Spin at 40C for 10 min with a speed of 2000 rpm-5000 rpm | + | |
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#Dissolve DNA pellet in 1 ml of TE containing 200 ug/ml of RNase, let stand at 370C for 30 minutes. | #Dissolve DNA pellet in 1 ml of TE containing 200 ug/ml of RNase, let stand at 370C for 30 minutes. | ||
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#Extract RNase by adding 1ml of TE-saturated phenol-chloroform, Then add 50 ul of 5 M NaCl and 2 ml of 100% ethanol to precipitate DNA , centrifuge at v 8000 rpm for 10 min. | #Extract RNase by adding 1ml of TE-saturated phenol-chloroform, Then add 50 ul of 5 M NaCl and 2 ml of 100% ethanol to precipitate DNA , centrifuge at v 8000 rpm for 10 min. | ||
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#Wash the DNA pellet with 70% ethanol, dissolve DNA in 500 ul of 0.1x TE buffer. Determine the DNA concentration and store DNA at – 200 C. | #Wash the DNA pellet with 70% ethanol, dissolve DNA in 500 ul of 0.1x TE buffer. Determine the DNA concentration and store DNA at – 200 C. | ||
Latest revision as of 00:20, 4 October 2012
Isolation of Genomic DNA from Gram-positive Bacteria
Saito and Miura 1963 (translated by Jie)
- Grow bacteria in 50 ml LB with antibiotic to late log phase or stationary phase, harvest cells at 40C, and centrifuge at 4000 rpm for 10 minutes.
- Resuspend cell pellet in 5 ml of Solution I (50mM glucose, 25 mM Tris-HCl, pH8.0, 10 mM EDTA).Add lysozyme to a final concentration of 5 mg/ml, shake at 370C for 30 minutes.
- Add 600 ul of 10% SDS and 300 ul of proteinase K (stock concentration 20 mg/ml) to the above mixture and let stand at 550C for 1 hour.
- Extract DNA by adding 5 ml of TE-saturated phenol-chloroform mix well and spin at 4000 rpm for 10 minutes or longer to get a good separation. Transfer the aqueous to a clean tube and repeat the same extraction step two more times.
- Transfer the aqueous phase to a clean tube and add 250 ul of 5M NaCl and 10 ml of 100% ethanol to precipitate DNA. Spin at 40C for 10 min with a speed of 2000 rpm-5000 rpm.
- Dissolve DNA pellet in 1 ml of TE containing 200 ug/ml of RNase, let stand at 370C for 30 minutes.
- Extract RNase by adding 1ml of TE-saturated phenol-chloroform, Then add 50 ul of 5 M NaCl and 2 ml of 100% ethanol to precipitate DNA , centrifuge at v 8000 rpm for 10 min.
- Wash the DNA pellet with 70% ethanol, dissolve DNA in 500 ul of 0.1x TE buffer. Determine the DNA concentration and store DNA at – 200 C.
