Golden Gate Cloning - DNA Cloning and Assembly Methods - page 124

Cloning of the level −1 fragments can be performed using
commercial kits such as the pGEM-T (Promega), pJET (Fermentas),
and the TOPO ® TA (Invitrogen) kits. PCR products can also be
cloned effi ciently using blunt-end cloning with a protocol that uses
restriction-ligation [ 13 , 14 ]. The vector chosen for cloning of level −1
fragments should fulfi ll two requirements: (1) it should preferably
not contain any restriction site for the type IIS enzyme that will be
used for the following assembly step, i.e., BpiI in the present exam-
ple ( see Note 4 ), and (2) the vector backbone should have a differ-
ent selection marker from the destination vector used for the next
step of assembly (level 0 modules have a spectinomycin resistance
marker; therefore, level −1 cloning vectors can have an ampicillin
resistance marker). Since many cloning vectors have a BsaI restric-
tion site in the ampicillin resistance gene (for example, pGEM-T or
pJET or pUC19), we have made a modifi ed pUC19 vector that
lacks this site ( see Note 5 ).

1. Add 0.5 μ L of vector (50 ng), 1 μ L of PCR product (50-100 ng),

2 μ L of 10× ligation buffer, 1 μ L of SmaI enzyme (10 U), 1 μ L

of T4 DNA ligase (3 U), and 14.5 μ L of water (total volume of

20 L) into a tube. The reaction mix is incubated for 1-2 h at

room temperature or in a 25 °C incubator ( see Note 6 ).

μ

2. The entire ligation mix is transformed into DH10B chemically
competent cells and plated on LB plates containing X-gal and
the appropriate antibiotic.
3. White colonies (or sometimes pale blue when small inserts
are cloned) are picked and inoculated in 5 mL of LB medium
containing the appropriate antibiotic.
4. Plasmid DNA is extracted using the NucleoSpin ® Plasmid
Quick Pure kit following the manufacturer's instructions.
5. Plasmid DNA can be checked by restriction enzyme digestion
using BpiI, followed by analysis of the digested DNA by
agarose gel electrophoresis.
6. DNA from two minipreps is sent for sequencing using primers
M13RP and/or M13FP.
7. When a correct sequence has been identifi ed, DNA concentra-
tion of the plasmid prep is measured using the NanoDrop
ND2000.

3.5 Construction of
the Destination Vector

A destination vector compatible with the entry modules needs to
be made. The vector should contain two BpiI sites compatible with
the two fusion sites present at the beginning of the fi rst level −1
fragment and the end of the last fragment ( see Figs. 2 and 3 ). The
vector backbone should not contain any additional BpiI restriction
site and should have an antibiotic resistance gene different from