anyone can give me protocol of Dna extraction of cell line by manual method without using LDT and EDT buffer? In the place of EDT and LDT which buffer is used? in manual method
Regards.
As you know the process for obtain DNA has two steps. The first one is the lysis of the cells and the second one is the extraction of DNA.
For lysis buffer you can use:
- saccharose
- magnesium chloride
- triton 100X
- Tris HCl [1M] pH 7.5
For extraction buffer:
- Tris HCL 100 mM pH 8
- EDTA 50 mM pH 8
- NaCl 500 mM
- b-mercaptoethanol
- Polyvinylpyrrolidone
Hello Shivangi
i agree with Artur that the old fashioned manual method which Is referenced in maniatis is the way I still use and indeed originally learnt from Tom Maniatis book
This involves lysis of your cells in a hypotonic solution of Tris; sodium chloride and EDTA. This ruptures your cells by osmosis. Into that so called TNE buffer you put proteinase K which digests liberated cellular proteins including DNAses and thus provides a stable extraction envirinment
You can simply by the way add this TNE buffer + prot K directly to your cells in culture; leave for 5 minutes at room temp and then aliquot out the resulting very viscous lysate or for cells in suspension spin your cells down at 1000 - 2000rpm for 10 min and lyse the resulting pellet
Alternatively for adherent cells you could trypsanise for 2-5 min in 0.1% trypsin in PBS; spin the cell pellet down and then lyse as described
i have used both methods successfully over the years
With the lysate as mentioned by Artur you can then extract with neutral Tris buffered phenol and Chloroform followed by ethanol precipitation and wash of your pellet with 70% ethanol or use of neutral trizol instead of phenol. Alternatively you can mix your lysate with an equal volume of 70% ethanol and purify using many of the silica based column protocols supplied by Qiagen Sigma Promega etc
Artur has provided you with an invaluable reference in the form of Tom Maniatis but if you still require specific protocols for what I have said in general terms I can supply these when I return to the lab on Monday. Good luck !
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