Design and Construction of Multigenic Constructs for Plant Biotechnology Using the GoldenBraid Cloning Strategy - DNA Cloning and Assembly Methods - page 137

Table 1
GB Extensions for the three main categories in multigenic assemblies

Category Forward Reverse

Promoter (including 35s:YFC
and 35s:YFN)

5 ′ GG GGTCTC A GGAG -
GSP 3 ′

5 ′ GG GGTCTC A AATC -
GSP 3 ′

CDS (including FUL and SOC) 5 ′ GG GGTCTC A AATG -

GSP 3 ′

5 ′ GG GGTCTC A AAGC -
GSP 3 ′

Terminator (including T35s) 5 ′ GG GGTCTC A GCTT -

GSP 3

5 ′ GG GGTCTC A AGCG -

GSP 3

′ ′

BsaI recognition and cutting sites (corresponding to GB overhangs) are marked in bold and italics , respectively. GSP
gene-specifi c primers

3. The process for removing internal type IIS sites is depicted in
Fig. 2a where an internal BsmBI site from FUL is eliminated,
following a standard overlap extension PCR protocol
(OE-PCR). In the case of one internal site, two pairs of prim-
ers are required. Design the two external primers (FUL.F1
and FUL.R2 in Fig. 2a ) as described above (Subheading 3.1 ,
step 1 , using extensions shown in Table 1 ). Design the second
(internal) pair of primers (FUL.R1 and FUL.F2) incorporat-
ing a nucleotide mismatch so as to mutate the internal Type
IIS BsmBI site. Keep in mind that for CDSs, the open reading
frame should be maintained (see primers designed for FUL in
Table 2 ). The internal primers must overlap at least 20 nt.
Perform the OE-PCR as follows:

(a) Prepare the fi rst pair of reactions using primer pairs FUL.
F1-FUL.R1 and FUL.F2-FUL.R2 ( see Note 2 ) and a
suitable template. Run an electrophoresis gel to verify the
success of the PCR (use only 1/5 of the volume) and
purify the rest of the reaction using QIAquick PCR
Purifi cation Kit ( see Note 3 ).

(b) Prepare the overlapping reaction with an equimolar ratio of
both PCR fragments as template. Do not add any primers
at the beginning. Set your PCR in two different steps:

Step 1 : 10 cycles with an annealing temperature determined
by the overlapping region between the two fragments
( see Note 4 ).

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Step 2 : Add 10 M of the external primers (FUL.F1 and

FUL.R2). Set the reaction for 25 cycles with the anneal-
ing temperature determined by the external pair of
primers. At the end, check the PCR by agarose gel
electrophoresis (Fig. 2c , Lane 5). Once it is correct,
proceed to step 6 .

μ

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