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Solid-phase cloning for high-throughput assembly of single and multiple DNA parts.

Lundqvist M, Edfors F, Sivertsson Å, Hallström BM, Hudson EP, Tegel H, Holmberg A, Uhlén M, Rockberg J - Nucleic Acids Res. (2015)

Bottom Line: We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors.In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in.The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%.

View Article: PubMed Central - PubMed

Affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.

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Unit-operations for RE-based and head-to-tail SPC. The light gray boxes indicate operations performed inside and yellow boxes indicate operations performed outside liquid handlers. Pink boxes are operations requiring temperature blocks while blue boxes indicate operations carried out in room temperature. (A) Restriction enzyme based SPC: PCR amplified inserts are automatically purified digested and subsequently ligated into a predigested vector. (B) Head-to-tail SPC: PCR amplified inserts and backbones are automatically purified, sequentially assembled by overlap extension and circularized into a plasmid. Transformation of Escherichia coli is done outside of the robot for both protocols.
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Figure 4: Unit-operations for RE-based and head-to-tail SPC. The light gray boxes indicate operations performed inside and yellow boxes indicate operations performed outside liquid handlers. Pink boxes are operations requiring temperature blocks while blue boxes indicate operations carried out in room temperature. (A) Restriction enzyme based SPC: PCR amplified inserts are automatically purified digested and subsequently ligated into a predigested vector. (B) Head-to-tail SPC: PCR amplified inserts and backbones are automatically purified, sequentially assembled by overlap extension and circularized into a plasmid. Transformation of Escherichia coli is done outside of the robot for both protocols.

Mentions: Ten different two-fragment constructs of various lengths were assembled (Table 1. 1–10, in Figure 5C, bands 4 and 7–11 are examples of these). Supplementary Table S2 presents a more detailed view of Table 1, including insert lengths, number of picked colonies and success rate. All constructs, generated colonies with correct product. The average success rate per colony was 87% and the largest construct assembled was 7.2 kbp. The protocol was adapted for automated cloning using a liquid handling robot and several key features was shared with the RE-based SPC (Figure 4A and B). Both methods make use of liquid handlers with a temperature block and magnetic capture capabilities. RE-based SPC was set up on a laboratory workstation (see ‘Materials and Methods’ section for details) to prepare cloning inserts for 96 clones in parallel. With existing head-to-tail protocols, 12 constructs can be made in parallel assembling up to four DNA fragments on the Magnatrix 1200 workstation or up to 96 constructs of two fragments on Magnatrix 8000+ workstation. These can easily be reprogrammed to make more constructs or constructs with different number of inserts depending on application. The standard protocol for head-to-tail SPC takes 2 h and 30 min to go from PCR products to ligated plasmids and the 96-construct assembly takes 6 h. Detailed flow-charts for adaptation to other workstations can be found in the supplementary material (Supplementary Figure S1).


Solid-phase cloning for high-throughput assembly of single and multiple DNA parts.

Lundqvist M, Edfors F, Sivertsson Å, Hallström BM, Hudson EP, Tegel H, Holmberg A, Uhlén M, Rockberg J - Nucleic Acids Res. (2015)

Unit-operations for RE-based and head-to-tail SPC. The light gray boxes indicate operations performed inside and yellow boxes indicate operations performed outside liquid handlers. Pink boxes are operations requiring temperature blocks while blue boxes indicate operations carried out in room temperature. (A) Restriction enzyme based SPC: PCR amplified inserts are automatically purified digested and subsequently ligated into a predigested vector. (B) Head-to-tail SPC: PCR amplified inserts and backbones are automatically purified, sequentially assembled by overlap extension and circularized into a plasmid. Transformation of Escherichia coli is done outside of the robot for both protocols.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4402512&req=5

Figure 4: Unit-operations for RE-based and head-to-tail SPC. The light gray boxes indicate operations performed inside and yellow boxes indicate operations performed outside liquid handlers. Pink boxes are operations requiring temperature blocks while blue boxes indicate operations carried out in room temperature. (A) Restriction enzyme based SPC: PCR amplified inserts are automatically purified digested and subsequently ligated into a predigested vector. (B) Head-to-tail SPC: PCR amplified inserts and backbones are automatically purified, sequentially assembled by overlap extension and circularized into a plasmid. Transformation of Escherichia coli is done outside of the robot for both protocols.
Mentions: Ten different two-fragment constructs of various lengths were assembled (Table 1. 1–10, in Figure 5C, bands 4 and 7–11 are examples of these). Supplementary Table S2 presents a more detailed view of Table 1, including insert lengths, number of picked colonies and success rate. All constructs, generated colonies with correct product. The average success rate per colony was 87% and the largest construct assembled was 7.2 kbp. The protocol was adapted for automated cloning using a liquid handling robot and several key features was shared with the RE-based SPC (Figure 4A and B). Both methods make use of liquid handlers with a temperature block and magnetic capture capabilities. RE-based SPC was set up on a laboratory workstation (see ‘Materials and Methods’ section for details) to prepare cloning inserts for 96 clones in parallel. With existing head-to-tail protocols, 12 constructs can be made in parallel assembling up to four DNA fragments on the Magnatrix 1200 workstation or up to 96 constructs of two fragments on Magnatrix 8000+ workstation. These can easily be reprogrammed to make more constructs or constructs with different number of inserts depending on application. The standard protocol for head-to-tail SPC takes 2 h and 30 min to go from PCR products to ligated plasmids and the 96-construct assembly takes 6 h. Detailed flow-charts for adaptation to other workstations can be found in the supplementary material (Supplementary Figure S1).

Bottom Line: We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors.In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in.The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%.

View Article: PubMed Central - PubMed

Affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.

Show MeSH
Related in: MedlinePlus