I am using Dan Swarr and Dave Frank's protocol to ligate oligos to the PX459 vector, using Stbl3 cells (invitrogen). However, I get very few colonies (max 3) after transformation using 20ul of cells and 2ul of ligation. Can I exchange Stbl3 cells with MACH1 or DH5alpha cells? Do you think there might be a problem in the cells or in the ligation step?
Thank you for your gentle attention.
Regards,
Celina
From my experience DH5alpha cells give good results. I also used Top10 (https://www.thermofisher.com/order/catalog/product/C404006) and Stellar cells. I am not sure what exactly your project is but sometimes the problem could be the preparation of the competent cells, the vector etc.
You can test your cells by transforming it with your empty vector or if you do not prepare the cell so the company usually send a control plasmid ( such as pUC19 or pUC56) with the competent cells. if you do that and still get a small number of colonies then your cells are old or died for so many reasons including incorrect storage. Stbl3 is a very good competent cell especially if your insert is unstable and transformation efficiency is greater than 1 × 108 cfu/µg plasmid DNA (>10-fold higher than alternative strains such as TOP10).
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