FastDigest SfaAI

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Prior to the USER reaction, the parental vectors were digested with FastDigest AsiSI (Thermo Fisher Scientific) and nicked with Nb. BsmI (New England Biolabs). The USER reactions were transformed into chemically competent E. coli DH5α. Correct assembly was verified by sequencing.

Protocol tips
Prior to the USER reaction, the parental vectors were digested with FastDigest AsiSI (Thermo Fisher Scientific) and nicked with Nb. BsmI (New England Biolabs). The USER reactions were transformed into chemically competent E. coli DH5α. Correct assembly was verified by sequencing.

Publication protocol

"Plasmid construction
The genes encoding phytoene synthase/lycopene cyclase (crtYB), phytoene desaturase (crtI) and geranylgeranyl diphosphate synthase (crtE) from X. dendrorhous were obtained from Addgene [14]. Genes encoding X. dendrorhous astaxanthin synthase crtS (GenBank accession number AX034665) and cytochrome P450 reductase crtR (GeneBank accession number EU884134), Paracoccus sp. N81106 β-carotene ketolase crtW (GenBank accession number AB206672) and P. ananatis β-carotene hydrolase crtZ (GenBank accession number D90087) were codon-optimized for Y. lipolytica and synthesized as GeneArt String DNA fragments by Thermo Fisher Scientific.

The plasmids, BioBricks, and primers used in this study are listed in Supplementary Table 3, 4, and 5, respectively. BioBricks were amplified by PCR using Phusion U polymerase (Thermo Fisher Scientific) under the following conditions: 98 °C for 30 s; 6 cycles of 98 °C for 10 s, 51 °C for 20 s and 72 °C for 30 s/kb; 26 cycles of 98 °C for 10 s, 58 °C for 20 s and 72 °C for 30 s/kb, and 72 °C for 5 min. BioBricks were purified from agarose gels using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel). BioBricks were assembled by into EasyCloneYALI vectors using USER cloning [15].

BioBricks were incubated in CutSmart® buffer (New England Biolabs) with USER enzyme and the parental vector for 25 min at 37 °C, followed by 10 min at 25 °C, 10 min at 20 °C and 10 min at 15 °C. Prior to the USER reaction, the parental vectors were digested with FastDigest AsiSI (Thermo Fisher Scientific) and nicked with Nb. BsmI (New England Biolabs). The USER reactions were transformed into chemically competent E. coli DH5α. Correct assembly was verified by sequencing."

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Restriction Enzymes AsiSI / SfaAI / SgfI using FastDigest SfaAI from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for FastDigest SfaAI below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Restriction Enzymes AsiSI / SfaAI / SgfI using FastDigest SfaAI from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.