Team:Lethbridge/Notebook/Protocols
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- | ===<font color="white">Maxiprep=== | + | ===<font color="white">Purification of Plasmid DNA by Alkaline Lysis (Large Scale AKA Maxiprep)=== |
<ol> | <ol> | ||
<li>Grow up a 500mL overnight culture in LB media containing the appropriate antibiotic corresponding to the restance of the plasmid.</li> | <li>Grow up a 500mL overnight culture in LB media containing the appropriate antibiotic corresponding to the restance of the plasmid.</li> | ||
<li>Centrifuge the cells at 5000 rpm for 10 minutes, discard the supernatant, transfer pellet to a 50mL falcon tube, record the weight of the pellet, and store at -20<sup>o</sup>C. ( 1- 2.5g cell pellets are expected)</li> | <li>Centrifuge the cells at 5000 rpm for 10 minutes, discard the supernatant, transfer pellet to a 50mL falcon tube, record the weight of the pellet, and store at -20<sup>o</sup>C. ( 1- 2.5g cell pellets are expected)</li> | ||
- | <li>Resuspend the cell pellet in 6mL Alkaline Lysis | + | <li>Resuspend the cell pellet in 6mL Alkaline Lysis Solution I (ALS1). Vortex carefully and slowly; may use a clean glass stir rod. Add 20µL of 1mg/mL RNase A.</li> |
<li>Add 1 mL of 10 mg/mL lysozyme (in 20mM Tris-HCl, pH 8.0)</li> | <li>Add 1 mL of 10 mg/mL lysozyme (in 20mM Tris-HCl, pH 8.0)</li> | ||
<li>Incubate at room temperature for 10 minutes.</li> | <li>Incubate at room temperature for 10 minutes.</li> | ||
<li>Add 12mL of <u>fresh</u> ALS2, mix well, do not vortex, and incubate on ice for 10 minutes.</li> | <li>Add 12mL of <u>fresh</u> ALS2, mix well, do not vortex, and incubate on ice for 10 minutes.</li> | ||
- | <li>Add 9mL of | + | <li>Add 9mL of ice cold ALS3, mix well, and incubate for 10 minutes on ice.</li> |
<li>Centrifuge at 4<sup>o</sup>C and 5000 rpm for 15 minutes.</li> | <li>Centrifuge at 4<sup>o</sup>C and 5000 rpm for 15 minutes.</li> | ||
- | <li>Decant supernatant filter within funnel into fresh 50mL | + | <li>Decant supernatant filter within funnel into fresh 50mL centrifuge tube.</li> |
<li><b>1:1 phenol:chloroform extraction:</b> In the fume hood, add 4mL of phenol/chloroform (1:1 -- 2mL of phenol + 2mL of chloroform), vortex for 15 seconds, centrifuge at 4000 rpm and 12<sup>o</sup>C for 4 minutes, and collect the upper aqueous phase. To the aqueous phase, add 4mL of chloroform, vortex for 15 seconds, centrifuge at 4000 rpm and 12<sup>o</sup>C for 4 minutes, and save the upper aqueous layer.</li> | <li><b>1:1 phenol:chloroform extraction:</b> In the fume hood, add 4mL of phenol/chloroform (1:1 -- 2mL of phenol + 2mL of chloroform), vortex for 15 seconds, centrifuge at 4000 rpm and 12<sup>o</sup>C for 4 minutes, and collect the upper aqueous phase. To the aqueous phase, add 4mL of chloroform, vortex for 15 seconds, centrifuge at 4000 rpm and 12<sup>o</sup>C for 4 minutes, and save the upper aqueous layer.</li> | ||
<li>Add 0.6 volumes of isopropanol to the saved aqueous layer and incubate on ice for 10 minutes. (Alternatively, precipitate overnight at -20<sup>o</sup>C)</li> | <li>Add 0.6 volumes of isopropanol to the saved aqueous layer and incubate on ice for 10 minutes. (Alternatively, precipitate overnight at -20<sup>o</sup>C)</li> | ||
<li>Centrifuge at 4<sup>o</sup>C and 5000 rpm for 15 minutes.</li> | <li>Centrifuge at 4<sup>o</sup>C and 5000 rpm for 15 minutes.</li> | ||
- | <li>Decant | + | <li>Decant supernatant into a fresh falcon tube (can be saved for further plasmid DNA isolation)</li> |
<li>Wash DNA pellet with 2mL 70% ethanol; centrifuge at 4<sup>o</sup>C and 5000 rpm for 5 minutes.</li> | <li>Wash DNA pellet with 2mL 70% ethanol; centrifuge at 4<sup>o</sup>C and 5000 rpm for 5 minutes.</li> | ||
<li>Air dry the DNA pellet; resuspend in 4mL 20mM Tris-HCl pH 8.0 by vortexing.</li> | <li>Air dry the DNA pellet; resuspend in 4mL 20mM Tris-HCl pH 8.0 by vortexing.</li> |