Welcome to the forums at seaphages.org. Please feel free to ask any questions related to the SEA-PHAGES program. Any logged-in user may post new topics and reply to existing topics. If you'd like to see a new forum created, please contact us using our form or email us at info@seaphages.org.
Recent Activity
Viknesh Sivanathan posted in did you know you can do restriction digests in the microwave?
nic.vega posted in did you know you can do restriction digests in the microwave?
nic.vega posted in did you know you can do restriction digests in the microwave?
Viknesh Sivanathan posted in did you know you can do restriction digests in the microwave?
nic.vega posted in did you know you can do restriction digests in the microwave?
All posts created by Poxleitner
| Link to this post | posted 17 Nov, 2015 23:33 | |
|---|---|
|
Hello, In my experience the low yields are not directly related to the nucleases, but to issues with the guanadinium to open the capsid. The heating really seems to combat this, but you have to inactivate the nucleases with the EDTA first. In addition, I think the advice to dilute high titers was specific for the phage precipitation protocol. We find that we can not get good yields with any titers less than 10^9. I hope this helps! Marianne |
| Link to this post | posted 17 Nov, 2015 18:44 | |
|---|---|
|
|
Hello, I have never diluted any of my lysates prior to extracting DNA using the new protocol. In my experience, the higher the lysate, the higher the yield. If you are getting really low yields you can try adding two steps. First, you can add 15 uL of 0.5 molar EDTA to the sample following the 30 minute nuclease incubation. This will inactivate most of the nucleases. Then you can incubate the lysate at 65 degrees for 15 minutes to start degrading the capsid. Then you can proceed with the protocol and add the DNA clean up resin. I hope this helps! Sincerely, Marianne |