I received an shRNA plasmid of 7.5kb blotted onto a paper. The spot was cut out & dissolved in 200uL nuclease-free water and transformed 5uL into DH5-alpha competent cells, but found no colonies. Competent cells(made by Cacl2 method) worked fine with a control plasmid of 6kb. Assuming the comp cells to be ok, a low amount of plasmid DNA was suspected to be the reason for transformation failure. So, the amount of plasmid DNA was measured using Qubit Fluorimetry assay (Invitrogen) and found to be 0.1ng/uL. How can this plasmid be efficiently transformed to get at least few colonies?
Use Stellar Competent Cells from Clontech which works with pg quantity of plasmid DNA. I have used it before for 13.5 kb plasmid.
Thanks for your reply. But, we have noticed that the competency of any bacterial strain is drastically low when we receive them after shipment. I work in India, and the shipment delays we experience here are really quite long. Is there any lab recipe/method which can be modified to work more efficiently?
A more efficient transformation method is the Hanahan method (see Sambrook et al for the method). It's far more efficient.
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