Design and Construction of Multigenic Constructs for Plant Biotechnology Using the GoldenBraid Cloning Strategy - DNA Cloning and Assembly Methods - page 139

Table 2
GB Oligonucleotides for the amplifi cation of the GBparts used in this chapter

GB part Primer Sequence

35s:YFN and
35s:YFC

35s.F1 5 ′ GGGGTCTCAGGAG ACTAGAGCCAAGCTGATCTC 3 ′

Linker 5 ′ GGGGTCTCACATT AGCGATCCACCTCCACCAGAT 3 ′

SOC SOC.F1 5 ′ GGGGTCTCAAATG GTGAGGGGCAAAACTCA 3 ′

SOC.R1 5 ′ GGGGTCTCAAAGC TCACTTTCTTGAAGAACAAGGTAAC

′

CCAATGAACAATTGTGTCTCTACTTCAGAAC 3

FUL FUL.F1 5 ′ GGGGTCTCAAATG GGAAGAGGTAGGGTTCA 3 ′

FUL.R1 5 ′ CAAACAACTTGTTGGCCG C GACGTTTCACAAAGTG 3 ′

FUL.F2 5 ′ TTTCACTTTGTGAAACGTC G CGGCCAACAAGTTG 3 ′

FUL.R2 5 ′ GGGGTCTCAAAGC TCACTCGTTCGTAGTGGTAGGAC 3 ′

T35s T35s.F1 5 ′ GGGGTCTCAGCTT CGGCCATGCTAGAGTCCGCAAA 3 ′

T35s.R1 5 ′ GGGGTCTCAAGCG AGGTCACTGGATTTTGGTTT 3 ′

GB Extensions are marked in bold . 35s:YFN and 35s:YFC are amplifi ed using the same pair of oligos as these oligos
bind to the 35s and to the linker located after the YFP half

4. If the internal restriction site is close enough to the 5

ends of the GBpart (see example for the GB part SOC1 in
Fig. 2b ), the situation is solved by making the GB oligo longer,
and introducing a mutation in the recognition sequence of the
restriction enzyme. Keep in mind that in the case of a CDS, the
open reading frame should be maintained. Proceed to step 5 .
5. PCR the GBpart using a suitable template and specially
designed GB primers. Verify the correct amplifi cation by aga-
rose gel electrophoresis. Primers used for the amplifi cations are
listed in Table 2 (Fig. 2c , Lanes 2, 3, 4, and 6).
6. Purify the PCR products using QIAquick PCR Purifi cation Kit
( see Note 5 ).
7. Add 3

′ or 3 ′

A overhangs with Taq DNA Polymerase. Set the reac-
tion by mixing 17

′

μ L of the purifi ed PCR, 2 μ L 10 × reaction

buffer, 0.5 μ L dNTPs, and 0.5 μ L Taq Polymerase.

Fig. 2 (continued) second step, an overlapping PCR using both PRC products as templates and using only FUL.
F1 and FUL.R2 primers is performed. The fi nal GB-FUL PCR lacks the internal BsmBI site. ( b ) Removal of a Type
IIS site close to the 3 end of SOC1 by mutating the site on the reverse primer of the GBpart, making it longer

than usual. ( c ) Agarose gel electrophoresis of the PCRs for the 5 GBparts described. Lane 1 : DNA Marker; Lane
2 : 35s:YFN; Lane 3 : 35s:YFC; Lane 4 : SOC1; Lane 5 : FUL; Lane 6 : T35s; Lane 7 : DNA Marker
( d ) BsaI digestion of the 5 GB parts generated. Each of them has three bands, two of them from pGEMT (1,622
and 1,433 pb) and a third one corresponding to the GB part. Lane 1 : DNA Marker; Lane 2 : pE35s:YFN; Lane 3 :
pE_35s:YFC; Lane 4 : pE_SOC1; Lane 5 : pE_FUL; Lane 6 : pE_T35s; Lane 7 : DNA Marker

′