12 Life Technologies
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| Vector Construction and Preparation
Vector Construction and Preparation
7. Clone second chain (for example, light chain of IgG) into the EcoRV/
PacI site of the vector containing the first-chain build, as described
above. The second-chain cDNA needs to be cut with a restriction
enzyme generating blunt ends or blunt-ended using T4 polymerase,
for example. Alternatively, engineer your gene so it is flanked with
EcoRV and PacI sites.
8. Perform colony PCR from the EcoRV/PacI cloning to confirm
presence of insert (Figure 3); be sure to sequence insert(s) before
proceeding with DNA preparation for transfection.
For successful cloning into the Freedom
®
pCHO 1.0 Vector:
1. Make sure total DNA in E. coli transformation does not exceed
recommended amount (<100 ng total DNA).
2. Make sure insert is in 3x molar excess over vector.
3. Make sure transformations are plated to kanamycin-containing plates
(50 µg/mL).
Tip:
