We recently transformation pNL4,3 E-R- Luc+ plasmid(about 16kb) with Trans-1 competent cells(our lab made it). We picked ten more clone to perform digestion identification but all clones are negative. At first we suspected that the plasmid is contaminated, so we take a gel purification but it still failed. What's the major concern in transformation this plasdmid? The competent cell line should be a important issue?
It might be helpful to use a strain designed for large plasmids like this: https://www.neb.com/products/c3019-neb-10-beta-competent-e-coli-high-efficiency.
Also, check out this previous question:
https://www.researchgate.net/post/Why_did_our_plasmid_yield_16kb_which_was_transformed_into_bacteria_decrease_significantly_when_we_used_a_bacterial_glycerol_stock
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