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Ligafast

Manufactured by Promega
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LigaFast is a rapid ligation kit designed for fast and efficient DNA fragment assembly. It simplifies the ligation process by providing a ready-to-use reaction mix, eliminating the need for time-consuming preparation steps. LigaFast enables the rapid joining of DNA fragments, facilitating cloning and recombinant DNA techniques.

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Lab products found in correlation

Hiseq 2000, by Illumina (4 mentions) End it repair kit, by Illumina (3 mentions) Phusion dna polymerase, by New England Biolabs (3 mentions) Dynaloligo dt beads, by Thermo Fisher Scientific (2 mentions) Rna fragmentation reagent, by Thermo Fisher Scientific (2 mentions) Pe adapter oligo mix, by Illumina (2 mentions) Phusion dna polymerase master mix, by New England Biolabs (2 mentions) Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Mirneasy kit, by Qiagen (2 mentions) Klenow exo minus, by New England Biolabs (2 mentions) Bioruptor, by Diagenode (2 mentions) Dynabeads oligo dt 25, by Thermo Fisher Scientific (1 mentions) Klenow exo, by New England Biolabs (1 mentions) Genome analyzer iix, by Illumina (1 mentions) Pcr clean up system, by Promega (1 mentions) Ecori, by Promega (1 mentions)

6 protocols using ligafast

1

ChIP-seq and RIP-seq Library Preparation

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Libraries for ChIP-seq were prepared according to manufacturer's instructions (Illumina, San Diego, CA) and as described (Asp et al., 2011 (link)). Briefly, IP'ed DNA (∼5 ng) was end-repaired using End-It Repair Kit (Epicenter, Madison, WI), tailed with deoxyadenine using Klenow exo− (New England Biolabs), and ligated to custom adapters with LigaFast (Promega). Fragments of 300 ± 50 bp were size-selected and subjected to ligation-mediated PCR amplification using Phusion DNA polymerase (New England Biolabs). Libraries were quantified by qPCR using primers annealing to the adapter sequence and sequenced at a concentration of 7 pM on an Illumina Genome Analyzer IIx or 10 pM on an Illumina HiSeq. In some cases barcoding was utilized for multiplexing.
For RIP-seq libraries, polyA+ RNA was isolated using Dynabeads Oligo(dT)25 (Invitrogen) beads and constructed into strand-specific libraries using the dUTP method (Parkhomchuk et al., 2009 (link)). Once dUTP-marked double-stranded cDNA was obtained, the remaining library construction steps followed the same protocol as described above for ChIP-seq libraries.
Bonasio R., Lecona E., Narendra V., Voigt P., Parisi F., Kluger Y, & Reinberg D. (2014). Interactions with RNA direct the Polycomb group protein SCML2 to chromatin where it represses target genes. eLife, 3, e02637.
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2

iPSC-derived Cardiomyocyte Transcriptome Analysis

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Eight paired-end cDNA libraries (two biological replicates of proband iPSC-CMs, two biological replicates of father iPSC-CMs, one biological replicate of two sibling iPSC-CMs, and one biological replicate of two control iPSC-CMs) were prepared and sequenced. Total RNA was extracted and quantified using miRNeasy Kit (Qiagen) according to the manufacturer’s protocol. 10 μg of total RNA was used to generate index-tagged paired-end cDNA libraries. Briefly, mRNAs were purified by polyA enrichment procedure using Dynal Oligo(dT) beads (Life technologies). mRNA fragmentation was performed using RNA Fragmentation Reagents (Life technologies) to obtain 200–300 bp fragments. cDNA was generated using SuperScript Double-Stranded cDNA Synthesis Kit (Life technologies). Illumina sequencing adapters were ligated to cDNA using LigaFast (Promega) and PE Adapter Oligo Mix (Illumina, San Diego, CA). PCR was performed on the adapter-ligated cDNA with 2X Phusion DNA polymerase Master Mix (New England Biolabs, Ipswich, MA). Sequencing was performed with Illumina’s HiSeq2000 or 2500 platform using paired end reads at an average length of 100 bps (2×100).
Kodo K., Ong S.G., Jahanbani F., Termglinchan V., Hirono K., InanlooRahatloo K., Ebert A.D., Shukla P., Abilez O.J., Churko J.M., Karakikes I., Jung G., Ichida F., Wu S.M., Snyder M.P., Bernstein D, & Wu J.C. (2016). iPSC-derived cardiomyocytes reveal abnormal TGFβ signaling in left ventricular non-compaction cardiomyopathy. Nature cell biology, 18(10), 1031-1042.
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3

Chromatin Immunoprecipitation (ChIP) Protocol

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Chromatin IP (ChIP) was performed as described previously16 (link). Briefly, crosslinked and isolated nuclei were sonicated using a Diagenode Bioruptor to an average size of ~250 bp. After pre-clearing with BSA-blocked protein G Sepharose, chromatin was incubated with antibodies at 4°C overnight. The chromatin immunocomplexes were recovered with the same BSA-blocked protein G beads. For ChIP-seq library construction, 5–10 ng of DNA extracted from the chromatin immunocomplexes as described previously15 (link). Libraries were prepared according to manufacturer's instructions (Illumina) and as described15 (link). Immunoprecipitated DNA was first end-repaired using End-It Repair Kit (Epicenter), tailed with an A using Klenow exo minus (NEB M0212), and ligated to custom adapters with LigaFast (Promega, city, state #M8225). Fragments of 350±50 bp were size-selected and subjected to ligation-mediated PCR amplification (LM-PCR), using Phusion DNA polymerase (NEB M0530). Libraries were quantified with quantitative PCR using primers annealing to the adapter sequence and sequenced at a concentration of 7 pM on an Illumina HiSeq 2000. All sequencing data has been deposited into GEO/NCBI with the accession number GSE60411.
Gao Z., Lee P., Stafford J.M., von Schimmelmann M., Schaefer A, & Reinberg D. (2014). AUTS2 confers gene activation to Polycomb group proteins in the CNS. Nature, 516(7531), 349-354.
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4

Cloning and Expression Vector Construction

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DNA manipulations and cloning were carried out using previously standardized procedures [58 ]. Once expanded, pUC57-LaccGluc-Stop and pUC57-LaccPost-Stop were digested simultaneously with EcoRI and NotI (Promega, Madison WI USA). Enzyme digestion products were evidenced in 1% TAE agarose gel, extracted and purified with and PCR Clean-Up System (Promega). Purified fragments were ligated using LigaFast (Promega) to constitutive expression vector pGAPZαA that was previously digested with EcoRI and NotI (Promega) resistant to ZeocinTM. DH5α E. coli was transformed with ligation products, and selected for their capacity to grown in LB media supplemented with Zeocin at 40 µg mL-1. Plasmid DNA extraction was carried out by using Miniprep Purification System (Promega). Expression vectors were identified as pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop. To demonstrate insert presence construct pGAPZαA-LaccGluc-Stop was digested with EcoRI and BamHI. pGAPZαA-LaccPost-Stop was digested with EcoRI, BamHI, and HindIII. All restriction enzymes were purchased from New England BioLabs (New England BioLabs, Ipswich MA USA).
Rivera-Hoyos C.M., Morales-Álvarez E.D., Poveda-Cuevas S.A., Reyes-Guzmán E.A., Poutou-Piñales R.A., Reyes-Montaño E.A., Pedroza-Rodríguez A.M., Rodríguez-Vázquez R, & Cardozo-Bernal Á.M. (2015). Computational Analysis and Low-Scale Constitutive Expression of Laccases Synthetic Genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris. PLoS ONE, 10(1), e0116524.
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5

Chromatin Immunoprecipitation (ChIP) Protocol

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Chromatin IP (ChIP) was performed as described previously16 (link). Briefly, crosslinked and isolated nuclei were sonicated using a Diagenode Bioruptor to an average size of ~250 bp. After pre-clearing with BSA-blocked protein G Sepharose, chromatin was incubated with antibodies at 4°C overnight. The chromatin immunocomplexes were recovered with the same BSA-blocked protein G beads. For ChIP-seq library construction, 5–10 ng of DNA extracted from the chromatin immunocomplexes as described previously15 (link). Libraries were prepared according to manufacturer's instructions (Illumina) and as described15 (link). Immunoprecipitated DNA was first end-repaired using End-It Repair Kit (Epicenter), tailed with an A using Klenow exo minus (NEB M0212), and ligated to custom adapters with LigaFast (Promega, city, state #M8225). Fragments of 350±50 bp were size-selected and subjected to ligation-mediated PCR amplification (LM-PCR), using Phusion DNA polymerase (NEB M0530). Libraries were quantified with quantitative PCR using primers annealing to the adapter sequence and sequenced at a concentration of 7 pM on an Illumina HiSeq 2000. All sequencing data has been deposited into GEO/NCBI with the accession number GSE60411.
Gao Z., Lee P., Stafford J.M., von Schimmelmann M., Schaefer A, & Reinberg D. (2014). AUTS2 confers gene activation to Polycomb group proteins in the CNS. Nature, 516(7531), 349-354.
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6

iPSC-derived Cardiomyocyte Transcriptome Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Eight paired-end cDNA libraries (two biological replicates of proband iPSC-CMs, two biological replicates of father iPSC-CMs, one biological replicate of two sibling iPSC-CMs, and one biological replicate of two control iPSC-CMs) were prepared and sequenced. Total RNA was extracted and quantified using miRNeasy Kit (Qiagen) according to the manufacturer’s protocol. 10 μg of total RNA was used to generate index-tagged paired-end cDNA libraries. Briefly, mRNAs were purified by polyA enrichment procedure using Dynal Oligo(dT) beads (Life technologies). mRNA fragmentation was performed using RNA Fragmentation Reagents (Life technologies) to obtain 200–300 bp fragments. cDNA was generated using SuperScript Double-Stranded cDNA Synthesis Kit (Life technologies). Illumina sequencing adapters were ligated to cDNA using LigaFast (Promega) and PE Adapter Oligo Mix (Illumina, San Diego, CA). PCR was performed on the adapter-ligated cDNA with 2X Phusion DNA polymerase Master Mix (New England Biolabs, Ipswich, MA). Sequencing was performed with Illumina’s HiSeq2000 or 2500 platform using paired end reads at an average length of 100 bps (2×100).
Kodo K., Ong S.G., Jahanbani F., Termglinchan V., Hirono K., InanlooRahatloo K., Ebert A.D., Shukla P., Abilez O.J., Churko J.M., Karakikes I., Jung G., Ichida F., Wu S.M., Snyder M.P., Bernstein D, & Wu J.C. (2016). iPSC-derived cardiomyocytes reveal abnormal TGFβ signaling in left ventricular non-compaction cardiomyopathy. Nature cell biology, 18(10), 1031-1042.
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