Hi all,
I am new to PFGE and I was wondering if anyone has done them specifically on B. thailandensis. I am having trouble with the ramped pulsed times. Additionally, I am not getting any bands with my standard markers and when I cut B. thailandensis with several enzymes. Any suggestions or comments would be helpful! Thanks.
Dear Kasandra, we perfom PFGE with different types of bacteria. In your gel first there can be a problem in plug preparation. Second, possible nuclease contamination in running buffer or pfge electrophoresis system. You should clean system. DNA remains in plugs in your gel photo.
Mert Sudagidan, thank you for your helpful feedback. I was going to clean the system before I ran another experiment; hopefully with the combination of cleaning the system and trying to troubleshoot other areas I can get this right.
To update anyone following this thread:
The parameters I followed are as stated -- 1.0% (w/v) agarose in 0.5X TBE; 200 V (6V/cm) with a switch time of 50 sec to 90 sec at 14°C for 24 hours.
Additionally, the standard was switched to the lambda ladder (Bio-Rad, cat. 1703635).
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