Dear Colleagues,
I am planning to detect and discover DNA viruses particularly those with a circular genome and transmitted by insect vectors. However, I am confused about selecting the best method for viral DNA extractions from insects. Your suggestions will be highly appreciated.
Adnan
Recently I extracted baculovirus shuttle vector (circular dsDNA 136 kb) from DH10b cells using the plasmid miniprep kit. However, instead of using filter columns I used the old isopropanol method to purify the isolated DNA.
Dear Dr Saber,
Thanks for your answering and I would like to figure out your protocol. Which concentration of Isopropanol have you used? Also, the plasmid miniprep kit from which company did you use?
Thanks in advance
I used Thermo plasmid miniprep kit.
- Grow 10ml bacterial culture and harvest the pellet
- Follow the kit instruction using resuspension, lysis and neutralization solutions.
- Instead of using columns and proceeding to wash step, precipitate the DNA using isopropanol.
- Add isopropanol (1:1) and mix it by inverting the tube for several times.
-Keep it on ice for 20 min.
-Centrifuge (13000 rpm for 15 min)
-Discard the supernatant
-Wash the pellet adding 500 ul EtOH 70%
-Centrifuge (13000 rpm for 5 min)
-Discard the EtOH and air dry the pellet
-Elute the pellet in 40 ul water
Good luck!
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