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Self-assembly (SA) cloning procedure. The PCR amplification reaction was performed using PrimeSTAR Max DNA polymerase (Takara Bio, Tokyo, Japan).
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Supplementary Material For:

Self-assembly cloning: a rapid construction method for recombinant molecules from multiple fragments Akira Matsumoto1 and Taichi Q. Itoh2 1 Department of Biology, Juntendo University School of Medicine, Inzai, Chiba, Japan and 2Graduate School of Systems Life Sciences, Kyushu University, Hakozaki, Fukuoka, Japan and Research Fellow of the Japan Society for the Promotion of Science, Japan BioTechniques 51:55-56 ( July 2011) doi 10.2144/000113705 Keywords: ligase-free cloning; self-assembly; multiple fragments ligation; in-frame fusion gene Supplementary material for this article is available at www.BioTechniques.com/article/113705

Self-assembly (SA) cloning procedure

The reaction mixture was subjected to 30 cycles of 98°C for 10 s, 60°C for 5 s, and 72°C for 5 s per 1 kb of each fragment. Four PCRs, two for the vector and two for the insert, were performed using the enzymefree cloning (EFC) procedure to clone one fragment into one vector (Supplementary Figure S1). After amplification, 1 µL DpnI (20 U; New England Biolabs, Ipswich, MA, USA) was added and incubated at 37°C for 60 min to digest template DNA. Then each reaction was purified and eluted into 30 µL distilled water by QIAquick gel extraction

The PCR amplification reaction was performed using PrimeSTAR Max DNA polymerase (Takara Bio, Tokyo, Japan) according to the manufacturer’s instructions. In brief, 1 ng template was amplified in a 50-µL reaction mixture by a Biometra T-Personal Thermal Cycler 48 (Biometra GmbH, Göttingen, Germany) with a compatible set of tailed and untailed primers, each of which was 10 pmol in the reaction.

primer 3

2

primer 1’

1

5’

primer 1

insert

3’

3’

5’

5’

3’

prim

3’

insert

primer 2

er

primer 3

1’

4

primer 4’

5’ primer 2’

2’

PCR

1

3’

5’

2

3’

3’

1’

5’

3’

2’

5’

5’

2

3’ 5’

1

2’

5’

1’

purification of PCR products 5’ 3’ 5’

1

3’ 3’

2

5’ 2’

5’

1’

3’

5’ overhang fragment

3’ 5’

1’

3’

3’

2

5’

1

3’

5’

3’

2’

5’

3’ overhang fragment

5’ overhang fragment

3’ overhang fragment

self-assembly 2

1

1’

2’

2

1

1’

2’

transformation

Supplementary Figure S1. Schematic diagram of SA cloning. To clone one fragment into a vector, four PCRs are performed: two parallel PCRs to amplify the insert and two for the vector. In each reaction, one primer is tailed with a specific sequence needed for subsequent SA. Sequences shown in box 1 and 2 complement those in box 1′ and 2′, respectively. Two parallel PCR products purified after digestion of templates by DpnI are mixed, denatured, and reannealed to produce 5′ and 3′ overhang fragments for the insert and vector. Finally, the insert and vector reactions are mixed, self-assembled, and transformed into competent cells.

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Templates and primers

In the primary experiments, we used pGL3-Basic (Promega Japan, Tokyo, Japan) as a template for the vector, and lacZ cDNA with the gpt promoter cloned into pSV-βGalactosidase (Promega) as a template for the insert. To build the multifunctional plasmid (Figure 2 in the main text), we used pAc5.1B (Invitrogen), pIZT/V5-His (Invitogen), and pDsRed1-C1 (Clontech Japan, Tokyo, Japan) as templates. All primers used in this study are listed in Supplementary Tables S1, S2, and S4.

Transfection of insect culture cells

3’

digestion of template by DpnI

denaturation & re-annealing

kit (QIAGEN K. K., Tokyo, Japan) and quantified by DU70 spectrophotometer (Beckmann, München, Germany). The denaturation and hybridization reaction was performed to produce fragments with singlestranded overhangs using 500 ng pooled two sibling-amplicons, one tailed on the 5′ end and the other on the 3′ end, in 20 µL hybridization buffer (100 mM Tris-HCl, 500 mM KCl, and 15 mM MgCl2 , pH 8.3). Two reactions, one for the vector and the other for the insert, were denatured at 95°C for 3 min followed by reannealing at 25°C for 3 min. Then, equal amounts of these reactions were mixed at 25°C for 5 min, and 7 µL reaction mixture were used to transform Escherichia coli One Shot TOP10 competent cell [1 × 108 colony formation units (cfu)/µg pUC19; Invitrogen Japan, Tokyo, Japan]. Transformed bacteria were cultured on the plate containing X-gal and ampicillin. We also used this SA cloning protocol for the multiple fragment reaction in this study, and transformed bacteria were cultured on the plate containing ampicillin, zeocin, and kanamycin.

Cultured Drosophila S2 cells on 24-well plates were transfected by using Effectene Transfection reagent (QIAGEN GmbH), as previously described (1), with 400 ng of the plasmid illustrated in Figure 2 of the main text. Fluorescence images were taken 24–48 h after transfection using Nikon Eclipse TS-100 inverted microscope equipped with a Digital Sight DS-L2 charge-coupled device (CCD) camera (Nikon, Tokyo, Japan).

References

1. Matsumoto, A., M. Ukai-Tadenuma, R.G. Yamada, J. Houl, K.D. Uno, T. Kasukawa, B. Dauwalder, T.Q. Itoh, et al. 2007. A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock. Genes Dev. 21:1687-1700.

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Benchmarks

Supplementary Table S1. Primer sequences. Primer number

Primer name

Sequence To amplify lacZ

primer1

Bgal-S1+0

5′-AGCACCATGGCCTGAAATAACCTC-3′

primer1′a

Bgal-S2+4

5′-GACAAGCACCATGGCCTGAAATAAC-3′

Bgal-S2+6

5′-GAGACAAGCACCATGGCCTGAAATAAC-3′

Bgal-S2+8

5′-GGAGACATAGCACCATGGCCTGAAATAAC-3′

Bgal-S2+10

5′-CTGGAGACATAGCACCATGGCCTGAAATAA-3′

Bgal-S2+12

5′-GTCTGGAGACATAGCACCATGGCCTGAAAT-3′

primer2

Bgal-A1+0

5′-AAAACGGGAAGTAGGCTCCCATGA-3′

primer2′a

Bgal-A2+4

5′-GGAAAAAACGGGAAGTAGGCTCCCA-3′

Bgal-A2+6

5′-GGAAAGAAAACGGGAAGTAGGCTCCCA-3′

Bgal-A2+8

5′-GGGAAAGTAAAACGGGAAGTAGGCTCCCA-3′

Bgal-A2+10

5′-GTGGGAAAGTAAAACGGGAAGTAGGCTCCC-3′

Bgal-A2+12

5′-GAGTGGGAAAGTAAAACGGGAAGTAGGCTC-3′

To amplify pGL3-Basic primer3

pGL3-4631S1+0

5′-TTCCGGTACGGGAGGTACTTGGAG-3′

primer3′a

pGL3-4631S1+4

5′-CTTTCCGGTACGGGAGGTACTTGGAG-3′

pGL3-4631S1+6

5′-ACTTTCCCGGTACGGGAGGTACTTGGAG-3′

pGL3-4631S1+8

5′-ACTTTCCCACGGTACGGGAGGTACTTGGAG-3′

pGL3-4631S1+10

5′-ACTTTCCCACGGTACGGGAGGTACTTGGAG-3′

pGL3-4631S1+12

5′-ACTTTCCCACTCGGTACGGGAGGTACTTGG-3′

primer4

pGL3-4619A1+0

5′-TACTTATCATGGTAGCTTGG-3′

primer4′a

pGL3-4619A1+4

5′-TGTCTACTTATCATGGTAGCTTGG-3′

pGL3-4619A1+6

5′-TGTCTCTACTTATCATGGTAGCTTGG-3′

pGL3-4619A1+8

5′-ATGTCTCCTACTTATCATGGTAGCTTGG-3′

pGL3-4619A1+10

5′-ATGTCTCCAGTACTTATCATGGTAGCTTGG-3′

pGL3-4619A1+12

5′-ATGTCTCCAGACTACTTATCATGGTAGCTT-3′

For primer number, see Supplementary Figure S1. The tailed sequences for single-stranded overhangs are represented in italics. a GC content of the overhang region of each primer was designed as 50%.

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Benchmarks

Supplementary Table S2. Mutated primer sequences. Primer number

Primer name

Sequence Primers for control reaction

a

To amplify lacZ primer1′

Bgal-S2

5′-GGAGACATTATTCCAGAAGTAGTGAGGA-3′

primer2

Bgal-A1

5′-GAAATACGGGCAGACATGGC-3′

primer1

Bgal-S1

5′-TATTCCAGAAGTAGTGAGGA-3′

primer2′

Bgal-A2

5′-GGGAAAGTGAAATACGGGCAGACATGGC-3′

To amplify pGL3-Basic primer3′

pGL3-4631S2

primer4

pGL3-4619A1

5′-TACTTATCATGGTAGCTTGG-3′

primer3

pGL3-4631S1

5′-GGTACGGGAGGTACTTGGAG-3′

primer4′

pGL3-4619A2

5′-ACTTTCCCGGTACGGGAGGTACTTGGAG-3′

5′-ATGTCTCCTACTTATCATGGTAGCTTGG-3′

Mutated primers

b

For a single mismatch in 5′ end primer1′

Bgal-S2-ver2

5′-cGAGACATTATTCCAGAAGTAGTGAGGA-3′

For a single mutation in 3′ end primer1′

Bgal-S2-ver3

5′-GGAGACAaTATTCCAGAAGTAGTGAGGA-3′

For a single mutation in center primer1′

Bgal-S2-ver4

5′-GGAcACATTATTCCAGAAGTAGTGAGGA-3′

For doubly mismatches both in 5′ end primer1′

Bgal-S2-ver5

5′-ccAGACATTATTCCAGAAGTAGTGAGGA-3′

For doubly mismatches in 5′ end and center primer1′

Bgal-S2-ver6

primer1′

Bgal-S2-ver10

5′-cGAcACATTATTCCAGAAGTAGTGAGGA-3′

For a single insertion in 5′ end 5′-cGGAGACATTATTCCAGAAGTAGTGAGGA-3′

For a single insertion in 3′ end primer1′

Bgal-S2-ver9

5′-GGAGACATcTATTCCAGAAGTAGTGAGGA-3′

For a single insertion in center primer1′

Bgal-S2-ver11

5′-GGAGcACATTATTCCAGAAGTAGTGAGGA-3′

Primer to change GC content of one overhangc For high GC content overhangs primer4′

pGL3-4619A2 ver7

5′-gTGcCTCCTACTTATCATGGTAGCTTGG-3′

primer1′

Bgal-S2-ver7

5′-GGAGgCAcTATTCCAGAAGTAGTGAGGA-3′

primer4′

pGL3-4619A2 ver8

primer1′

Bgal-S2-ver8

For low GC content overhangs 5′-ATaTCTtCTACTTATCATGGTAGCTTGG-3′ 5′-GaAGAtATTATTCCAGAAGTAGTGAGGA-3′

For primer number, see Supplementary Figure S1. The tailed sequences for single-stranded overhangs are represented in italics. a GC content in the tail regions of the primers was designed to be 50%. b Each primer was used instead of Bgal-S2 to synthesize lacZ fragment with a mutated overhang. The mutated nucleotide is represented as a small letter. c Each primer was used instead of the corresponding primer of its same name. The mutated nucleotides are represented as a small letter.

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Benchmarks

Supplementary Table S3. Cloning efficiency of SA cloning with two fragments having mutation in one cohesive end of eight nucleotides in length. Mutation in one single-stranded overhang

Relative rate of successful cloning (%)

Control (exact match, 50% of GC content)

100

Single mutation (about 50% of GC content) Mismatch in 5′ end

21.6

3′ end

10.1

middle

0.8

insertion in 5′ end

10.2

3′ end

13.3

middle

0.0

Double mutations (about 50% of GC content) 1.0

mismatches in 5′ end

0.0

5′ end + middle Different GC content 75%

49.3

25%

5.7

Supplementary Table S4. Primer sequences to build the multifunctional plasmid. Primer

Sequence To amplify fragment A

V5-S2-ECF2

5′-ACTTTCCCGGAAGGTAAGCCTATCCCTAA-3′

pAc5.1-forA

5′-CTGATGGAGCGGCTTTGTGTC-3′

V5-S2

5′-GAAGGTAAGCCTATCCCTAA-3′

pAc5.1-forA-ECF1

5′-ATGTCTCC CTGATGGAGCGGCTTTGTGTC-3′

pIZT 1975S EFC1

5′-GGAGACATCTGCAGCACGTGTTGACAAT-3′

pIZT Zeo stop-1 A

5′-GTCCTGCTCCTCGGCCACGAA-3′

pIZT 1975S

5′-CTGCAGCACGTGTTGACAAT-3′

NLS pIZT Zeo stop-1 Aa

5′-cttacgtttctttttGTCCTGCTCCTCGGCCACGAA-3′

NLS pIZT Zeo stop Sa

5′-aaaaagaaacgtaagTGACCGACGCCGACCAACACC-3′

pIZT 556A

5′-TTCGAACAGATGCTGTTCAACTG-3′

pIZT Zeo stop S

5′-TGACCGACGCCGACCAACACC-3′

pIZT 556A koz

5′-GGTGGCGATTCGAACAGATGCTGTTCAACTG-3′

DsRed 613S koz

5′-TCGCCACCATGGTGCGCTCCTCCAAGAAC-3′

DsRed-C1 3960 A

5′-GGGGTCTGACGCTCAGTGGAAC-3′

DsRed 613S

5′-ATGGTGCGCTCCTCCAAGAAC-3′

DsRed-C1 3960 EFC2 A

5′-CGGGAAAGTGGGGTCTGACGCTCAGTGGAAC-3′

To amplify fragment B

To amplify fragment C

To amplify fragment D

For the name of fragment, see Figure 2 in the main text. The tailed sequences for single-stranded overhangs are represented in italics. a The nucleotides to create NLS are represented as a small letter.

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