MIT7_13f08_lab16_Protocol_QiagenHiSpeed

MIT7_13f08_lab16_Protocol_QiagenHiSpeed - MIT OpenCourseWare

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MIT OpenCourseWare http://ocw.mit.edu 7.13 Experimental Microbial Genetics Fall 2008 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms .
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7.13 Fall 2008 Page | 1 Plasmid or Cosmid DNA Purification Using HiSpeed Plasmid Midi and Maxi Kits This protocol is for preparation of up to 200 μ g of high- or low-copy plasmid or cosmid DNA using the HiSpeed Plasmid Midi Kit or 750 μ g using the HiSpeed Plasmid Maxi Kit . A final DNA concentration of up to 0.4 μ g/ μ l (Midi) or 1.5 μ g/ μ l (Maxi) can be expected, if eluting a high-copy plasmid with 500 μ l of Buffer TE. * For high-copy plasmids, expected yields are 100–200 μ g for the HiSpeed Plasmid Midi Kit and 300–750 μ g for the HiSpeed Plasmid Maxi Kit. For low-copy plasmids, expected yields are 30–100 μ g for the HiSpeed Plasmid Midi Kit and 50–250 μ g for the HiSpeed Plasmid Maxi Kit using these culture volumes. Important notes before starting New users are advised to familiarize themselves with the detailed protocol provided in the HiSpeed Plasmid Purification Handbook posted at the 7.13 Stellar website. In addition, extensive background information is provided on the Qiagen plasmid resource page: www.qiagen.com/goto/plasmidinfo . Optional: Remove samples at the steps indicated with the symbol in order to monitor the procedure on an analytical gel (see page 29 of the HiSpeed Plasmid Purification Handbook for analytical procedure) Blue (marked with a ) denotes values for the HiSpeed Plasmid Midi Kit ; red (marked with a ) denotes values for the HiSpeed Plasmid Maxi Kit . Things to do before starting Add the provided RNase A solution to Buffer P1 before use (check for P1 + RNaseA in the fridge first). Use one vial of RNaseA (centrifuge briefly before use) per bottle of Buffer P1, to give a final concentration of 100 μ g/ml. Check Buffer P2 for SDS precipitation due to low storage temperatures. If necessary, dissolve the SDS by warming to 37°C. Pre-chill Buffer P3 at 4°C. Optional: Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use one vial LyseBlue (centrifuge briefly before use) per bottle of Buffer P1 to achieve a 1:1000 dilution (Check to see if LyseBlue has already been added). LyseBlue provides visual identification of optimum buffer mixing thereby preventing the common handling errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA, and cell debris. For more details see “Using LyseBlue reagent” on page 16 of the HiSpeed Plasmid Purification Handbook.
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7.13 Fall 2008 Page | 2 Procedure 1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate for approx. 8 h at 37°C with vigorous shaking (approx. 300 rpm).
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This note was uploaded on 11/11/2011 for the course BIO 7.012 taught by Professor Lander during the Fall '10 term at MIT.

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MIT7_13f08_lab16_Protocol_QiagenHiSpeed - MIT OpenCourseWare

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