3 replies to this topic

[ahfat83]

Hey guys,

I've been using this Lentiviral plasmid built on pLV backbone with hPGK drived GFP and EFS promoter for my insert GOI. The size is around 10Kb
When I do miniprep, the yield was OK though not as consistent as other plasmid.
(the reading on Nanodrop often didn't match with EtBr gel. The curve on the graph was normal,
but the signal in EtBr gel was much weaker than expected)
When growing in larger culture for midi/maxi. there is hardly any plasmid in the 100ml culture,
and I'm sure there were plasmid in the 2ml culture before I pour some of it into the larger Amp+ TB medium.
I also noticed the transformed E.coli colonies are much smaller comparing to other plasmid.

I'm not new to DNA prep and this never happen when I work on other plasmids like pLKO, pCMVsport, pBMGneo, pEF-BOS, pMD.G, pdelta8.91.
Anyone mind to share your experience tackling this particular annoying problem? Thanks!

Sean

[Bomber]

you may want to switch your bacteria.
occasionally it may happen that, as you also said, bacteria are affected by the plasmid and or its content.
it may help to transform a different bacteria strain to check if this handles the plasmid better..., worth a try mabye.

[Merrique]

Hi,
I have a similar problem. I have heard that the maxi kit used may be the problem. We use the normal Qiagen Maxi Kit. Is there any kit that can be recommended for lentiviral vectors?
Thanks,
Anna

[leochicaybam]

I sometimes have a recombination problem with my lentiviral vectors when growing them at 37ºC. Try to grow them at 30ºC.