What is the actual difference between extraction procedures of plasmid and Genomic DNA
The major difference in the extraction procedure for gDNA and pDNA is how we break open the cells to access the cargo and the inherent nature of the length as well as the integrity of both the genetic material. For gDNA, we blindly lyse the cells harshly (chemical or mechanical) to release all the contents and extract the gDNA by PCI separation. But, in the case of extracting Plasmids Alkaline Lysis method is followed wherein mild treatment to cell membrane breaks open the cells. Both the gDNA and the pDNA is denatured into a single strand by the SDS and NaOH in the lysis buffer. The reaction is further neutralized by the addition of any salts of acetate, wherein the plasmid DNA being smaller in size renatures easily, whereas, the gDNA being quite large and complicated remains denatured. In this regard, care should be taken, not to vortex harshly since the genomic DNA can further break and it's easy for small gDNA strands to renature and mix with the plasmid DNA containing aqueous phase.
Dear Sahoo, for the extraction of plasmid, we prefer commercially available kits from Qiagen or other companies. As explained by Dr. Gopalakrishnan Chandrasekaran, plasmid extraction a little bit more sensitive procedure than gDNA.
Best regards.
Dear saho, greetings, DNA isolation from Bacteria is so easy, please following this protocol step.
Materials Required:
Lysis buffer (40mM tris-acetate PH 7.8, 20mM sodium-acetate, 1mM EDTA, 1% SDS), chloroform, 5 MNacl, 70% EtOH, TE buffer.
Protocol:
1- Harvest bacteria colony into 1.5 ml Eppendorf tube.
2- resuspend cell pellet in 200 µl of lysis buffer (40 mM Tris-acetate PH 7.8, 20µl sodium – acetate, 1mM EDTA, 1% SDS) by vigorous pipetting.
3- Add 66 µl of 5M NaCl solution (to remove most protein and cell debris).
4- Centrifuge at 12000 rpm for 10 min at 4℃.
5- Transfer the clear supernatant into new vial, add equal volume of chloroform.
6- Shake tube gently at least 50 times until a milky solution completely formed.
7- Centrifuge 12000 rpm for 3min.
8- Transfer the supernatant to another vial
9- Add equal volume of cold absolute ethanol to precipitate DNA.
10-wash DNA with 70% Ethanol
11-dry DNA and dissolve in 50µl TE buffer or double distil water.
The yield of DNA around 50-150ug per 109 cells
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