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Biological and Medical Sciences > Classification by Discipline > Molecular Biology

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Branch: Protocol-Wiki Type: Protocol Intention: For Publication Read/Write Permissions: Open Access

Authors : 16.06.2010 - Christopher Dyer  -  Sci-Mate    09.06.2010 - John Stewart

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A super fast, easy and cheap method to prep plasmids for restriction enzyme digestion, sub-cloning or other molecular biological purposes.

[edit] GROW UP

1. Add 0.5 ml Super Broth + 0.5 ul Ampacillin (100mg/ml) + bacteria containing Amp resistant bacteria; spread plate with same innoculation. (of course, use whatever anti-biotic your plasid confers resistance to)
   
2. Shake 37⁰C overnight.
   

[edit] BOILING LYSIS

1. Centrifuge 1' (min) at 13 000 rpms.
   
2. Discard S/N by aspiration; resuspend pellet in 400 ul M-STET + 30ul lysozyme.
   
3. Vortex samples, then boil for 40-50 seconds. Centrifuge 5' at 13 000 rpms.
4. Use toothpick to remove protein pellet. Discard pellet.

[edit] ISOPROPANOL DNA PRECIPITATION

1. Add 20ul 3M NaAc (Ph5) + 300ul isopropanol. Mix; leave 10' at room temp.
2. Centrifuge 10' at 13 000; discard supernatant; rinse white or opaque DNA pellet with 70% ethanol; allow to dry.
3. Resuspend in 20-40 ul of TE. Be sure to wash sides of eppendorf to collect DNA.