Hi
I'm using qiagene kit to extract my plasmids DNA and recently I encountered problems to purify a lentivirus backbone plasmid (pMDLg/pRRE 8895 bp , Addgene=12251) from maxiprep.
I had no problems with miniprep. After I inoculated miniprep bacteria into big flask for large prep, bacteria grew well, every step looked fine but when I run DNA gel I found that there was only one band for uncut plasmid (looked like being linearized, but i did not add in any enzyme!) and there was smear in digested plasmid lane. To test whether my maxiprep kit is working or not, I have done maxiprep for another plasmid (pRSV-Rev 4174bp,pMD2.G 5824 bp) concurrently and it was alright for digestion of this plasmid after maxiprep, which means nothing went wrong in my maxiprep. I cannot figure out what the
problem is. I used same antibiotics (ampicilin), same LB, same maxiprep kit to prepare both constructs but I failed to produce the larger, lower yield plasmid. It is important to highlight that I have ever used the same kit to produce the lentivirus backbone plasmid for 3-4 times, I just encountered the problem of maxiprep in recent one month. So what makes my large prep plasmid being linearized/degraded during maxiprep? I have tried to pick more colonies from my amp plate and cultured with LB in small volume for miniprep and all clones are positive. I cannot understand why it cannot be purified with maxiprep kit? Anyway I hope I can solve this mystery asap with helps from you !!!
Thanks in advance :D
I agree with Paritosh. I have used the Qaigen kits (endo-free maxi) for AAV plasmid prep and been *very* disappointed with the yield. I ended up culturing 4 liters just to get the minimum of 300ug for virus production. Then I switched to phenol:chloroform:iaa purification with ice cold EtOH precipitation and made enough with just 50mL culture. Plus no expensive columns. Unfortunately it's important to make sure it's endo free (at least for AAV prep) if the next step is virus production or you'll kill the cells.
My suggestions are as follows:
1. You must use endotoxin free kit
2. After addition of P3 solution from the kit directly centrifuge without incubation
3. Add chloramphenicol to increase plasmid yield
@Liz Unger,
So how did you circumvent the endo-toxin problem while using the Phenol:Chloroform Method?
Best,
Arjun
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