Hi everyone, I am getting low yield for my retroviral plasmid i.e. ~50ng/ul. Also the bacterial colonies after the trasformation is very tiny and difficult to pick. Can anyone have any suggestion on how to get better yeild? I am using pBABE puro vector.
In the past I have used pCDH-CMV-puro, which also gave low yeold. Is the property of the retroviral plasmids?
Please post as soon as possible if you have any suggestions.
regards,
Ara
You may try low salt LB media to grow the culture for retro viral plasmid.
Try different salt concentration (low or high) and different temperature to grow the bacterial
In my experience, this is very dependent on which lentiviral or retroviral backbone and which E. coli strain you are using. I generally recommend JM109, and if this fails, then either HB101 or Stbl3. Some of these plasmids can be low copy number, if which case a chloramphenicol amplification step can greatly improve yields. Just make sure you catch the cultures in early log phase (OD 0.6-0.8), and supplement with glucose, when you add the chloramphenicol. These simple steps can improve plasmid yield 5-fold.
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