11. Evaporate leftover ethanol (37
C or RT) (see Note 6).
12. Dissolve all pellets in 10–40 μL ddH 2 O (pooling all ligation
reactions).
(a) First, pipette the droplet above the DNA pellet (the pellet
may be invisible).
Fig. 2 Workflow of library creation. Primers are designed to overlap with 4 bp
(green box) flanked by Bsa I sites (dark red box) on the 5
0
-side, so that the
overlap region will become single-stranded DNA after digestion. Several NNK
codons (red cross) can be introduced by a single primer pair. Amplifying the
target plasmid in a PCR yields a linear product (black) containing the mutations,
the Bsa I site on both sides of the duplicated 4 bp overlap. The template is
destroyed using Dpn I, single-strand overhangs created by Bsa I digest and the
product ligated
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C or RT) (see Note 6).
12. Dissolve all pellets in 10–40 μL ddH 2 O (pooling all ligation
reactions).
(a) First, pipette the droplet above the DNA pellet (the pellet
may be invisible).
Fig. 2 Workflow of library creation. Primers are designed to overlap with 4 bp
(green box) flanked by Bsa I sites (dark red box) on the 5
0
-side, so that the
overlap region will become single-stranded DNA after digestion. Several NNK
codons (red cross) can be introduced by a single primer pair. Amplifying the
target plasmid in a PCR yields a linear product (black) containing the mutations,
the Bsa I site on both sides of the duplicated 4 bp overlap. The template is
destroyed using Dpn I, single-strand overhangs created by Bsa I digest and the
product ligated
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Martin Spinck et al.