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Circligase buffer

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CircLigase Buffer is a buffer solution designed for use with the CircLigase enzyme, which is used in the circularization of single-stranded DNA or RNA molecules. The buffer provides the optimal conditions for the CircLigase enzyme to effectively catalyze the ligation of the ends of the nucleic acid molecules, forming a circular structure.

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Lab products found in correlation

Agencourt xp beads, by Beckman Coulter (5 mentions) Circligase 1, by Illumina (5 mentions)

Spelling variants of this product

CircLigase (22 citations) 10× CircLigase Buffer II (2 citations)

5 protocols using circligase buffer

1

cDNA Circularization and Purification

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To the cDNA, 3 μl 10× CircLigase Buffer (Epicentre), 1.5 μl 50 mM MnCl2, 1.5 μl 1 mM ATP, 0.5 μl 100 μM DNA adapter (oligonucleotide I; Supplementary Table S1), and 1 μl CircLigase I (Epicentre) were added. The reaction was incubated at 60°C for 2 h, then 80°C for 10 min to inactivate the ligase. The ligated DNA was EtOH precipitated with 20 mg glycogen as a carrier and dissolved in 20 μl of nuclease-free H2O. Then the cDNA was purified using 36 μl of Agencourt XP beads (Beckman Coulter), according to manufacturer's instructions and eluted with 20 μl TE buffer.
Watters K.E., Choudhary K., Aviran S., Lucks J.B., Perry K.L, & Thompson J.R. (2017). Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements. Nucleic Acids Research, 46(5), 2573-2584.
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2

Adapter Ligation for cDNA Library

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Adapter ligation was performed as described54 . Briefly, dissolved cDNA was mixed with 3 μl of 10x CircLigase Buffer (Epicentre), 1.5 μl of 50 mM MnCl2, 1.5 μl of 1 mM ATP 0.5 μl of 100 μM DNA adapter (Oligonucleotide M, Supplementary Table 4), and 1 μl of CircLigase I (Epicentre, Madison, WI), incubated at 60C for 2 h and 80C for 10 min. DNA was precipitated by adding 70 μl nuclease-free water, 10 μl 3M NaOAc pH 5.5, 1 μl 20 mg/ml glycogen, and 300 μl of 100% EtOH and storing at −80C for 30 min before centrifugation. Pellets were dissolved in 20 μl of nuclease-free water, purified using 36 μl of Agencourt XP beads (Beckman Coulter, Brea, CA) according to the manufacturer’s protocol, and eluted with 20 μl of 1X TE buffer.
Strobel E.J., Cheng L., Berman K.E., Carlson P.D, & Lucks J.B. (2019). A ligand gated strand displacement mechanism for ZTP riboswitch transcription control. Nature chemical biology, 15(11), 1067-1076.
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3

Adapter Ligation for cDNA Library

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Adapter ligation was performed as described54 . Briefly, dissolved cDNA was mixed with 3 μl of 10x CircLigase Buffer (Epicentre), 1.5 μl of 50 mM MnCl2, 1.5 μl of 1 mM ATP 0.5 μl of 100 μM DNA adapter (Oligonucleotide M, Supplementary Table 4), and 1 μl of CircLigase I (Epicentre, Madison, WI), incubated at 60C for 2 h and 80C for 10 min. DNA was precipitated by adding 70 μl nuclease-free water, 10 μl 3M NaOAc pH 5.5, 1 μl 20 mg/ml glycogen, and 300 μl of 100% EtOH and storing at −80C for 30 min before centrifugation. Pellets were dissolved in 20 μl of nuclease-free water, purified using 36 μl of Agencourt XP beads (Beckman Coulter, Brea, CA) according to the manufacturer’s protocol, and eluted with 20 μl of 1X TE buffer.
Strobel E.J., Cheng L., Berman K.E., Carlson P.D, & Lucks J.B. (2019). A ligand gated strand displacement mechanism for ZTP riboswitch transcription control. Nature chemical biology, 15(11), 1067-1076.
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4

cDNA Circularization and Purification

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To the cDNA, 3 µL 10X CircLigase Buffer (Epicentre), 1.5 µL 50 mM MnCl2, 1.5 µL 1 mM ATP, 0.5 µL 100 µM DNA adapter, oligonucleotide C (Supplementary Table 4), and 1 µL CircLigase I (Epicentre) were added. The reaction was incubated at 60 °C for 2 hr, then 80 °C for 10 min to inactivate the ligase. The ligated DNA was EtOH precipitated with 1 µL 20 mg/mL glycogen as a carrier and dissolved in 20 µL of nuclease-free H2O. Then the cDNA was purified using 36 µL of Agencourt XP beads (Beckman Coulter), according to manufacturer’s instructions and eluted with 20 µL TE buffer.
Watters K.E., Strobel E.J., Yu A.M., Lis J.T, & Lucks J.B. (2016). Cotranscriptional Folding of a Riboswitch at Nucleotide Resolution. Nature structural & molecular biology, 23(12), 1124-1131.
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5

cDNA Circularization and Purification

Check if the same lab product or an alternative is used in the 5 most similar protocols
To the cDNA, 3 µL 10X CircLigase Buffer (Epicentre), 1.5 µL 50 mM MnCl2, 1.5 µL 1 mM ATP, 0.5 µL 100 µM DNA adapter, oligonucleotide C (Supplementary Table 4), and 1 µL CircLigase I (Epicentre) were added. The reaction was incubated at 60 °C for 2 hr, then 80 °C for 10 min to inactivate the ligase. The ligated DNA was EtOH precipitated with 1 µL 20 mg/mL glycogen as a carrier and dissolved in 20 µL of nuclease-free H2O. Then the cDNA was purified using 36 µL of Agencourt XP beads (Beckman Coulter), according to manufacturer’s instructions and eluted with 20 µL TE buffer.
Watters K.E., Strobel E.J., Yu A.M., Lis J.T, & Lucks J.B. (2016). Cotranscriptional Folding of a Riboswitch at Nucleotide Resolution. Nature structural & molecular biology, 23(12), 1124-1131.
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