Don't:
Do:
8 Life Technologies
™
| Vector Construction and Preparation
Vector Construction and Preparation
Be sure to include with your gene of interest:
• Kozak sequence + ATG start codon
(for example: GCCACCATGG)
• Secretion leader sequence
• Stop codon
Optimize the gene using GeneArt
®
GeneOptimizer
®
services for CHO
codons, RNA secondary structure, cryptic splice sites, etc.
• Be sure to include your chosen signal peptide sequence in
this optimization
Sequence plasmid DNA from 2–3 colonies (from gene-plasmid junctions
through the entirety of the gene[s] of interest) using primers provided in
the User Guide (see Table 1).
Prepare high-quality, low-endotoxin DNA for transfection using an
appropriate plasmid DNA preparation kit (such as the PureLink
®
HiPure
Plasmid Midiprep or Maxiprep Kits).
Linearize DNA prior to transfection using NruI.
Optional: Remove NruI by phenol:chloroform extraction and
precipitation of DNA with alcohol.
• Using 5-Prime
™
Phase Lock Gel
™
barrier during phenol:chloroform
extraction is highly effective in preventing organic solvent carryover.
Avoid using the following (murine Ig heavy chain) signal peptide unless
the nucleotide sequence is optimized:
• ATG GGA TGG TCA TGT ATC ATC CTT TTT CTA GTA GCA ACT GCA
ACC GGT GTA CAT TCA
