Golden Gate Cloning - DNA Cloning and Assembly Methods - page 126

7. Incubate the plates overnight at 37 °C. Many white and very
few blue colonies should be obtained.
8. Pick a few white colonies from the plate ( see Note 10 ).
9. Check by restriction digest using BsaI (for MoClo level 0
modules) or any other suitable enzyme.

Level 0 modules that have been made from sequenced level −1
constructs do not need to be sequenced again. Modules that have
been directly assembled from PCR products of course need to be
sequenced. Level 0 modules are then ready for further assembly
using BsaI and ligase and MoClo vectors (described in ref. 10 ).

4 Notes

1. Two restriction sites located next to each other can be mutated
using a single long primer. Also, the fusion site may be selected
between the two sites than need to be mutated, and the two
mutations introduced by a single mismatch in each of the two
primers required at this fusion site. Therefore, the number of
PCR fragments that needs to be amplifi ed to remove n restric-
tion sites may sometimes be lower than n + 1.
2. KOD polymerase is a useful enzyme as it has proofreading
activity and does not add any nucleotide at the end of the
amplifi ed fragments (unlike Taq polymerase). Fragments ampli-
fi ed with KOD polymerase therefore have blunt ends, which is
a prerequisite for blunt-end cloning using SmaI.

3. Direct assembly of PCR products in a destination vector is
possible [ 15 ]. It is however recommended to purify the PCR
products using a column to remove DNA polymerase and
primer dimers. Indeed some of the primer dimers are fl anked
by two fusion sites (these are part of the primers) and can
therefore be cloned, resulting in incorrect constructs. The fi nal
constructs may also contain PCR-induced mutations and
therefore need to be sequenced.
4. The presence of a BpiI site in the vector backbone of level −1
modules would not prevent assembling them using Golden
Gate cloning, as the fi nal construct will not contain this vector
backbone. However, the presence of a BpiI site in all level −1
vector backbones would lead to continuous digestion and reli-
gation at this site, which would unnecessarily consume some
ATP from the ligation mix.
5. A simple strategy, enzymatic inverse PCR [ 16 ], can be used to
eliminate the internal BsaI site in pUC19. The entire plasmid
is amplifi ed with two primers designed to introduce a mutation
in the BsaI site: primers bsarem1 (ttt ggtctc a ggtt ctcgcggtat-
cattgcagc) and bsarem2 (ttt ggtctc a aacc acgctcaccggctccag).