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SBB12Ntbk-JonathanKotker: Difference between revisions
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→Jon Kotker, 1 March 2012
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'''Part pBca9525-sbb1204''': ''Miniprep Purification of DNA'' | '''Part pBca9525-sbb1204''': ''Miniprep Purification of DNA'' | ||
The procedure is described [http://openwetware.org/wiki/Template:SBB-Protocols_Micro3 here] and is used to extract and purify the DNA retrieved from the cells. | The procedure is described [http://openwetware.org/wiki/Template:SBB-Protocols_Micro3 here] and is used to extract and purify the DNA retrieved from the cells. It is replicated below for completeness. | ||
# Pellet 2mL saturated culture by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube. | |||
# Dump supernatant. | |||
# Add 250uL of P1 buffer into each tube. Resuspend the cells thoroughly using a vortexer. | |||
# Add 250uL of P2 buffer, a base that denatures everything and causes cells to lyse. Gently mix up and down. The solution should become clearer and blue. | |||
# Add 350uL of N3 buffer, an acid of pH ~5 that causes cell junk, including protein and chromosomal DNA, to precipitate, and leaves plasmids and other small molecules in solution. Slowly invert a few times, then shake. | |||
# Spin in centrifuge at top speed for 5 minutes. | |||
# Label blue columns with an alcohol-resistant lab pen. | |||
# Pour liquid into columns and place the columns into the centrifuge. Spin at full speed for 15 seconds. | |||
# Dump liquid out of the collectors under the columns. The DNA should be stuck to the white resin. | |||
# Wash each column with 500uL of PB buffer. | |||
# Spin in centrifuge at full speed for 15 seconds and flick out the liquid again. | |||
# Wash with 750uL of PE buffer, which washes the salts off the resins. | |||
# Spin in centrifuge at full speed for 15 seconds and flick out the liquid again. | |||
# Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol. | |||
# Label new Microcentrifuge tubes and put columns in them. | |||
# Elute them by adding 50uL of water down the middle of the column. Don't let it stick to the sides. | |||
# Spin in centrifuge at top speed for 30 seconds. | |||
# Take out the columns and cap the tubes. | |||
'''Part pBca9525-sbb1223''': ''Picking of Colonies'' | '''Part pBca9525-sbb1223''': ''Picking of Colonies'' | ||
