14 Life Technologies
™
| Vector Construction and Preparation
NruI is suggested as the vector linearization restriction enzyme
before stable pool generation. Is this enzyme (methylation sensitive)
compatible with TOP10 E. coli (dam-positive)?
Although TOP10 cells are not dam-negative (DNA produced in
these cells can be methylated), we have had no problem digesting
the Freedom
®
pCHO plasmid with NruI, as shown in the gel below
(Figure 4). PvuI may also be used for vector linearization should NruI
linearization be incomplete, or if an NruI site is brought in with your
gene(s) of interest.
Vector Construction and Preparation
1: Uncut pCHO-LC/HC
2: pCHO-LC/HC + NruI
M: 1 Kb Plus DNA ladder
Q:
A:
Figure 4. Gel confirming plasmid linearization; lane 1 (uncut) is supercoiled
DNA, which migrates faster than the NruI-linearized DNA in lane 2.
1 M 2
Kb
12
8
6
4
3
2
1
Expected size (bp) 15162
,
