DNA Extraction
In market there are many kits are present for DNA extraction in easy way but they are very expensive and Pakistan is a developing country they not afford such a expensive kits in our laboratory.So in Pakistan use these method this method much time consuming but not expensive DNA extraction make possible by organic method.
Following step for DNA extraction
Lysis
Purification
Precipitation
Washing
Elution
Lysis.
In first sample mist with the help of rod properly grinned meat of cat into eppendrof.If sample is liquid nitrogen store than air dry is necessary.if ethanol store than first to wash PBS than distil water and later air dry.
Take 20mg meat add 360 ul lysis and use Pk enzyme for preservation of DNA denaturation solution incubate the sample 56 degree for 6 to 8 hours Lysate are ready.
Purification.
Centrifuge the lysate at 10,000 rpm for 8 minutes take supernatant in to new tube add ethanol equal amount two layers appear one yellowish and upper clear DNA present in upper layer.pippet out upper layer in to new tube and add chloroform isoamyl 24;1 than again centrifuge 2 clear layer appear.
Precipitation.
Take upper layer shift in to new tube through pipett add 100% absolute ethanol centrifuge 8000 rpm for 8 minutes pellet are appear discard solution.
Washing.
In this step add 70% ethanol and NaACE 4% centrifuge 8000 rpm for 8 minutes and air dry the pellet.
Elution.
Add 40 ul T.E buffer 50% DNA extract.
http://feeds.feedburner.com/ecarepk