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Saturday, March 16, 2013

Purification of Plasmid DNA using PEG Precipitation

Minipreparations of plasmid DNA can be used as a templates in dideoxy sequencing reactions whose products can be analysed in automated machines. The length of the sequence established on run using the machines largely depends on the purity of plasmid minpreps, the addition of one or two steps in the procedure of plasmid miniprep by Alkaline Lysis by SDS method can increase the purity of plasmid by large fold, which will result in reproducible results with sequence length of more than 600bp on the final sequence run product.

Procedure for PEG Precipitation
  1. To 50 µL of miniprep plasmid add 8.0µL of 4M NaCl and 40µL of 13% PEG 8000 (w/v), incubate on ice for 20 – 30 mints.
  2. Centrifuge at maximum speed for 10mins at 4oC, collect the pellet by carefully removing the supernatant by gentle aspiration. 
  3. Rinse the pellet using 500µL of 70% Ethanol. The colour of the pellet changes to milky white at this stage. 
  4. Carefully remove the supernatant and rinse the pellet again with 500µL of 70% ethanol. 
  5. Remove the ethanol and keep the tube in inverted position till the trace off ethanol evaporates from the pellet. 
  6. Dissolve the pellet in 20 – 30 µL of sterile water. 
  7. Analyse the purity and integrity of the plasmid on agarose gel before submitting for sequencing. 
  8.  Plasmid with high purity and good quality is ready.