Hello

We're currently attempting to create a plasmid stock of lentivirus. We're using a pCK005_U6-Sp-sgRNA_UP-TdTomato_WPRE (#85453 addgene) in which we've removed the tdTomato and inserted a polylinker (Xba1, Ecor1) with a Cla1 restriction site to allow us to ligate in the inserts (all are with Ecor1, Cla1 ends). The miniprep (Turbo Birnboim and Doly - alkaline/SDS lysis and isopropanol precipitation) of this plasmid with both NEB stable and XL10-gold cells produced plasmid DNA that was correct - confirmed with restriction digest to release the insert and ran on EBr agarose gel. However, neither the midi or maxipreps ( Qiagen midi/zymo maxi) produce any yields of DNA - despite the bacteria growing normally. I'd greatly appreciate any suggestions about why this might be the case and whether we should try the classical chloro/phenol method?