Lidstrom:Miniprep

From OpenWetWare

Revision as of 21:29, 24 February 2012 by Janet B. Matsen (Talk | contribs)
Jump to: navigation, search

Back to Protocols

Tips You (Mostly) Won't Find in the Manual

  • Use 1 colony in 2 mL of TB +antibiotic grown overnight.
    • TB gives you a higher plasmid yield than LB, but our lab currently doesn't make it (Janet 2/23/12). If your plasmid is high-copy number, one tube is sufficient. If you have a medium copy plasmid, grow 2-3 cultures from the same colony in parallel and pool them before loading the column.
  • Elute in water, not the elution buffer that comes with the kit.
    • The elution buffer stabilizes DNA and reduces DNase activity, but comes at a cost -- the added chemicals are likely to lessen DNA sequencing and subsequent DNA manipulation successes.

Expected Yield

  • 2 mL of pSB1A3 (high copy) should yield ~ 150 ng/uL in 30 uL.
  • 2 2mL pSB3K3 (medium copy) cultures grown in parallel then pooled should yield ~ 30 ng/uL.

Miscelaneous

  • Our lab prefers the Fermentas miniprep kit over Quiagen, though I am not sure why. -Janet 2/23/12
  • Do not grow cultures with less than 4:1 air:liquid ratio -- it is very bad for plasmid prep. This is still one of Janet's open questions 2/23/12
  • You can elute in 30uL instead of 50uL to get a more concentrated sample.
  • Record the A260/A280 measurement down when you record the concentration. It should be between 1.8 and 2.
Personal tools
{"cc_re__cc_re__cc_by_sa_2_5": ["a href=\"/wiki/OpenWetWare:How_to_join\">\n\t\t\t<img style=\"margin:2px;\" \n\t\t\t\tsrc=\"http://openwetware.org/images/0/03/88x31_JoinOWW.png\" border=0 /></a>\n                </div>\n        </div>\n\t\t\t\t\t\t\t</div>\n\n\t\t\t</div><!-- end of the left column -->\n\t\t\n\t\t\n\t\t\t<div class=\"visualClear\"></div>\n\t\t\t<div id=\"footer\">\n\t\t\t\t<div id=\"f-poweredbyico\"><a href=\"http://www.mediawiki.org/\"><img src=\"/skins/common/images/poweredby_mediawiki_88x31.png\" alt=\"Powered by MediaWiki\" /></a></div>\n\t\t\t\t<div id=\"f-copyrightico\"><a href=\"http://creativecommons.org/licenses/by-sa/2.5/\"><img src=\"/somerights20.png\" alt='GNU FDL or Creative Commons BY-SA' /></a></div>\n\t\t\t<ul id=\"f-list\">\n\t\t\t\t<li id=\"copyright\">Content is available under <a href=\"/wiki/Copyright\" title=\"Copyright\">GNU FDL or Creative Commons BY-SA</a>.</li>\n\t\t\t\t<li id=\"privacy\"><a href=\"/wiki/OpenWetWare:Privacy_policy\" title=\"OpenWetWare:Privacy policy\">Privacy policy</a></li>\n\t\t\t\t<li id=\"about\"><a href=\"/wiki/OpenWetWare:About\" title=\"OpenWetWare:About\">About OpenWetWare</a></li>\n\t\t\t\t<li id=\"disclaimer\"><a href"], "copyright__copyright__copyright": ["n pooled should yield ~ 30 ng/uL.\n\nMiscelaneous\n\n  \u2022 Our lab prefers the Fermentas miniprep kit over Quiagen, though I am not sure why. -Janet 2/23/12\n  \u2022 Do not grow cultures with less than 4:1 air:liquid ratio -- it is very bad for plasmid prep. This is still one of Janet's open questions 2/23/12\n  \u2022 You can elute in 30uL instead of 50uL to get a more concentrated sample.\n  \u2022 Record the A260/A280 measurement down when you record the concentration. It should be between 1.8 and 2.\nPersonal tools", "n pooled should yield ~ 30 ng/uL.\n\nMiscelaneous\n\n  \u2022 Our lab prefers the Fermentas miniprep kit over Quiagen, though I am not sure why. -Janet 2/23/12\n  \u2022 Do not grow cultures with less than 4:1 air:liquid ratio -- it is very bad for plasmid prep. This is still one of Janet's open questions 2/23/12\n  \u2022 You can elute in 30uL instead of 50uL to get a more concentrated sample.\n  \u2022 Record the A260/A280 measurement down when you record the concentration. It should be between 1.8 and 2.\nPersonal tools", "n pooled should yield ~ 30 ng/uL.\n\nMiscelaneous\n\n  \u2022 Our lab prefers the Fermentas miniprep kit over Quiagen, though I am not sure why. -Janet 2/23/12\n  \u2022 Do not grow cultures with less than 4:1 air:liquid ratio -- it is very bad for plasmid prep. This is still one of Janet's open questions 2/23/12\n  \u2022 You can elute in 30uL instead of 50uL to get a more concentrated sample.\n  \u2022 Record the A260/A280 measurement down when you record the concentration. It should be between 1.8 and 2.\nPersonal tools", "n pooled should yield ~ 30 ng/uL.\n\nMiscelaneous\n\n  \u2022 Our lab prefers the Fermentas miniprep kit over Quiagen, though I am not sure why. -Janet 2/23/12\n  \u2022 Do not grow cultures with less than 4:1 air:liquid ratio -- it is very bad for plasmid prep. This is still one of Janet's open questions 2/23/12\n  \u2022 You can elute in 30uL instead of 50uL to get a more concentrated sample.\n  \u2022 Record the A260/A280 measurement down when you record the concentration. It should be between 1.8 and 2.\nPersonal tools", "n pooled should yield ~ 30 ng/uL.\n\nMiscelaneous\n\n  \u2022 Our lab prefers the Fermentas miniprep kit over Quiagen, though I am not sure why. -Janet 2/23/12\n  \u2022 Do not grow cultures with less than 4:1 air:liquid ratio -- it is very bad for plasmid prep. This is still one of Janet's open questions 2/23/12\n  \u2022 You can elute in 30uL instead of 50uL to get a more concentrated sample.\n  \u2022 Record the A260/A280 measurement down when you record the concentration. It should be between 1.8 and 2.\nPersonal tools", "n pooled should yield ~ 30 ng/uL.\n\nMiscelaneous\n\n  \u2022 Our lab prefers the Fermentas miniprep kit over Quiagen, though I am not sure why. -Janet 2/23/12\n  \u2022 Do not grow cultures with less than 4:1 air:liquid ratio -- it is very bad for plasmid prep. This is still one of Janet's open questions 2/23/12\n  \u2022 You can elute in 30uL instead of 50uL to get a more concentrated sample.\n  \u2022 Record the A260/A280 measurement down when you record the concentration. It should be between 1.8 and 2.\nPersonal tools"]}