Hello! Is it possible to use Phenol:Chloroform:Isoamyl Alcohol (25:24:1) to extract genomic DNA? how to ensure no RNA is present in the sample. If anyone has a protocol for this, I kindly ask if you´d mind sharing it.
Best regards,
Rita.
https://www.researchgate.net/post/how_to_prepare_phage_gnome_for_Next_seq#view=5b0b4ee91a5e76ae506a728b
Genome extraction of the phage was performed using phenol Chloroform Isoamyl Alcohol (PCI) method, with some modifications. The phage lysate containing 10^11 bacteriophages, was filtered with a sterile syringe filter having pore size 0.22µm (Merck Millipore), DNase, RNase and 12.5µl of 1M MgCl2 were added in 1 ml of the filtrate and incubated at 37oC for 4 hours. The DNases and RNases were denatured at 75oC for 10 minutes in water bath and then shifted to 55oC and 50µl 0.5M EDTA, 40 µl 10%SDS and 2.5µl of proteinase K (20mg/ml) were added and incubated at 37oC for 1 hour while mixing gently after every 20 Mins. After 1 hour 550µl of the lysate is transferred to 2 Eppendorf tubes each and equal amount of PCI is added and mixed gently by inverting the tubes. The tubes are then centrifuged at 13000rpm for 10 Mins. The upper layer is picked carefully, and transferred to the new Eppendorf tubes and ice chilled 95% ethanol (1ml) and 50µl of ice chilled 3M sodium acetate is added and kept on ice for 5 Mins to facilitate precipitation, centrifuged at 13000rpm for 10 Mins. Pellet is formed, the supernatant is thrown away and 500µl of 70% ethanol is added in each tube and spun at 13000rpm for 5 Mins. The supernatant is thrown away and the tubes are inverted on blotting paper for drying. After 15-20Mins, DNase/RNase free water is added to dissolve DNA pellet. The tubes are kept overnight at room temperature for maximum dissolution. The genome thus obtained was run on 1% agarose gel in 1% TAE Buffer in the presence of a Ladder and visualized in a Gel documentation system.
- Badges
- Science topic
- Similar topics
- Psychology
- Cognitive Science
- Mind