How to extract cyanobacterial genome
Leonardo Ken Okumura
Disclaimer
This experimental protocol is provided "as is" without warranty of any kind, either express or implied, including, but not limited to, the implied warranties of merchantability and fitness for a particular purpose. The entire risk as to the quality and performance of the protocol is with you. Should the protocol prove defective, you assume the cost of all necessary servicing, repair, or correction. In no event will the authors or any other party who distributed the protocol be liable for any damages, including any lost profits, lost monies, or other incidental or consequential damages arising out of the use or inability to use the protocol (including but not limited to loss of data or data being rendered inaccurate or losses sustained by you or third parties or a failure of the protocol to operate with any other programs).
Abstract
This protocol describes how to extract cyanobacterial genomes. The main treatments are carried out by sodium iodide, lysozyme, proteinase K and SDS.
Steps
Extracting cyanobacteria genome
Centrifuge the cyanobacteria culture at 16,000 x g for 10 minutes and remove the supernatant.
Add 400 µL of Extraction Buffer and 100 µL of Saturated NaI Solution to the pellet, and mix well.
Incubate at 37°C for 30 minutes.
Centrifuge at 177,000 x g for 5 minutes and remove the supernatant.
Add 100 µL of Extraction Buffer to the pellet and mix. Then centrifuge at 177,000 x g for 5 minutes and remove the supernatant.
Add 500 µL of Extraction Buffer with Lysozyme to the pellet and mix.
Incubate at 37°C for 2 hours.
Add 5 µL of Proteinase K Solution and 15 µL of SDS, and mix.
Incubate at 37°C overnight.
Add 125 µL of SDS, and mix.
Incubate at 37°C for more than 3 hours.
Add an equal volume of PCI and gently rotate for 5 minutes.
Centrifuge at 4,000 rpm for 10 minutes at room temperature, and transfer the supernatant to a new tube.
Add 3 µL of RNase A, and mix.
Incubate at 37°C for 30 minutes.
Repeat steps 12 and 13.
Add isopropanol in more than equal volume and mix.
Centrifuge at 16,000 x g for 1 minute at 4°C and remove the supernatant.
Repeat steps 17 and 18, replacing isopropanol with 70% ethanol.
Air-dry the pellet and resuspend in distilled water.
