3. Clean up digested PCR and vector DNA by gel or method of choice.
4. Set up ligations with at least 3x molar excess of PCR insert.
5. Transform One Shot
®
TOP10 E. coli cells and plate on kanamycin plates.
(Case study: After transformation of One Shot
®
TOP10 cells and colony
PCR, all 18 tested colonies contained an insert of the correct size
(Figure 2). )
6. We recommend sequencing 2–3 colonies before proceeding with
insertion of the second gene (Figure 1).
FAQ Sections
P
CMV
First insertion site
5'
3'
3' 5'
GCCACCATG
Gene of interest 1
Up Primer 1
Up Primer 2
Down Primer 2
Down Primer 1
STOP
P
CMV
Second insertion site
5'
3'
3' 5'
GCCACCATG
Gene of interest 2
STOP
Figure 1. In the figure above, the 5' ends of the primers have sequences for restriction enzyme
recognition sites that will be incorporated into the final PCR product for cloning into pCHO.
The extra sequence at the 5' ends of the primers will not base-pair with the initial gene
target(s); however, after several PCR cycles, the annealing temperature can be raised to
increase the specificity of the primers for the gene plus new flanking sequences.
11 Life Technologies
™
| Freedom
®
CHO-S
®
Kit FAQ
