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Help me--why my plasmid DNA is lost after digestion with Enzyme - (Jan/24/2006 )

Hi Dear all, blink.gif ohmy.gif blink.gif

These two days I met a very weird thing.

I am working on 3 lentivirus clones. Firstly I cloned my target gene in pENTR vector, and then did the LR reaction with plenti6-v5 destination vector. After selected wiht Ampicilin, blasticidin and chloramphenicol, I picked the clone that looks positive and then mini-prep the plasmid . After I finished the mini-prep, I run the gel to check the plasmid, they looks good as the plenti6-v5 destination vector prepared by plasmid maxi-prep. Kit.
The weird thing is that :
when I digested those plasmid from my clones as well as the plenti6-v5 destination vector, the plenti6-v5 destination vector was correctly digested and gave the right bands, but the plasmids from those clones never gave any DNA band, and all the DNA was gone. Some of them gave a lot of RNA in 200-300bp position, others are just empty, but when I run gel to check the plasmid, they definitly looks like plasmid, not bacteria genomic DNA. I also can be sure that my digestion(my enzyme, buffer, water and TE) has no problem(because other maxi-prep. plasmid digestion is OK)

I met these problem two times in woking this clones, each time I check around 20 clones, all of them have this weird problem. I just do not know what happens to my clones?
Does anybody have this kind experience?
Thanks in advance! rolleyes.gif rolleyes.gif



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-Taras Bulba-