Purified DNA (see previous section) was quantified by Fragment Analyzer (Agilent). 25 ng of purified DNA was resuspended up to 50 μl in water. 10 μl of T4 DNA ligase buffer (NEB, B0202), 4 μl of 10 mM dNTPs, 5 μl of T4 DNA polymerase (NEB, M0203), 1 μl of Klenow DNA polymerase (NEB, M0210), 5 μl of T4 DNA polynucleotide kinase (NEB, M0201), and 25 μl of water was added to the diluted input DNA and incubated at 25°C for 30 min. Samples were purified with Ampure XP beads (Beckman, A36881) and resuspended in 32 μl of water. 5 μl of buffer 2 (NEB, B7002), 1 μl of 10 mM dATP, 3 μl of Klenow fragment (NEB, M0212), and 9 μl of water was added to the end-repaired DNA and incubated at 37°C for 30 min. Samples were purified with Ampure XP beads (Beckman, A36881) and resuspended in 23 μl of water. 5 μl of Truseq Y adaptors for paired-end sequencing (custom-made), 5 μl of 10× ligase buffer (NEB, B0202), 1.5 μl of T4 DNA ligase (NEB, M0202), and 15.5 μl of water was added to the 3’-adenylated DNA and incubated at room temperature for 1 h. Samples were purified with Ampure XP beads (Beckman, A36881) and resuspended in 30 μl of water. 3 μl of adaptor ligated DNA was used for PCR amplification (KAPA HiFi master mix, KK201).
Ligase buffer
Manufactured by New England Biolabs
10× ligase buffer is a solution used in molecular biology applications to facilitate the ligation of DNA fragments. It provides the necessary conditions, such as optimal pH and ion concentrations, to enable the enzymatic joining of DNA ends by DNA ligase. The buffer's core function is to support the ligation reaction, enabling the formation of phosphodiester bonds between compatible DNA ends.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by New England Biolabs (5 mentions)
T4 dna ligase buffer, by New England Biolabs (3 mentions)
Fragment analyzer, by Agilent Technologies (2 mentions)
Klenow dna polymerase, by New England Biolabs (2 mentions)
T4 dna polynucleotide kinase, by New England Biolabs (2 mentions)
Klenow fragment, by New England Biolabs (2 mentions)
T4 dna polymerase, by New England Biolabs (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Thermocycler, by Bio-Rad (1 mentions)
Esp3i, by New England Biolabs (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
T4 pnk, by New England Biolabs (1 mentions)
Sanger sequencing, by Microsynth (1 mentions)
Failsafe enzyme mix, by Illumina (1 mentions)
Ecori, by New England Biolabs (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
6 protocols using ligase buffer
1
DNA Library Preparation for Sequencing
2
Lentiviral Vector Cloning Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The vector backbone (lentiGuide-Puro, Plasmid no. 52963, a gift from F. Zhang23 ) was digested with Esp3I (NEB) and treated with rSAP (NEB) at 37 °C for 3 h and gel-purified on a 0.5% agarose gel. For sgRNA phospho-annealing (sequences in Supplementary Note 4 ), 1 µL of sgRNA top and bottom strand oligonucleotide (100 µM each), 1 µL 10× T4 DNA Ligase Buffer, 1 µL T4 PNK (NEB) and 6 µL H2O were mixed and incubated in a thermocycler (BioRad) using the following program: 37 °C for 30 min, 95 °C for 5 min, ramp down to 25 °C at a rate of 5 °C/min. The annealed oligonucleotides were diluted 1:100 in H2O and ligated into the vector backbone using 50 ng digested lentiGuide-Puro plasmid, 1 µL annealed oligonucleotide, 1 µL 10× Ligase Buffer (NEB), and 1 µL T4 DNA Ligase (NEB) in a 10 µL reaction (filled up to total volume with H2O). The ligation mix was incubated at room temperature for 3 h and transformed into NEB Stable Competent E. coli (C3040H) following the manufacturer’s instructions. Correct assembly of the sgRNA into the backbone was confirmed by SANGER-Sequencing (Microsynth) and plasmids were isolated using a GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific) following the manufacturer’s instructions.
3
DNA Library Preparation for Sequencing
4
Genomic DNA Adapter Ligation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The adaptor was ligated to the digested genomic DNA overnight (16–20 h) at 16 °C. The ligation reaction consisted of 2.0 µL of adaptor, 4 µL of digested genomic DNA, 2 µL of 10× New England Biolabs ligase buffer, 1 µL of T4 DNA ligase (NEB #M0202S). The reaction was stopped by incubating the mixture at 80 °C for 20 min. Seventy µL of TE was added to the reaction before use as template during the PCR amplification.
5
Quantitative Telomere Length Measurement
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
STELA was performed as described previously (Baird et al. 2003 (link); Sfeir et al. 2005 (link)). Briefly, 2 µg of genomic DNA was digested with EcoRI (New England Biolabs) overnight at 37°C. Ten nanograms of digested DNA was ligated to telorettes in 10 µL of ligation buffer (5 U of T4 DNA ligase [New England Biolabs], 1× ligase buffer [New England Biolabs], 0.18 nM individual telorettes) overnight at 35°C. Ligated DNA (250 pg) was amplified by PCR in 25 µL of PCR mix (0.2 µM XpYpE2 primer, 0.2 µM teltail primer, 1× Fail Safe PCR buffer H, 2 U of Fail Safe enzyme mix [Epicentre] with 27 cycles [15 sec at 95°C, 20 sec at 58°C, and 9 min at 68°C]). PCR products were resolved on a 0.7% agarose/TAE gel, blotted onto Hybond (GE Healthcare), UV cross-linked in a Stratalinker, prehybridized with Church mix (0.5 M sodium phosphate buffer at pH 7.2, 1 mM EDTA, 0.7% SDS, 0.1% BSA), and hybridized overnight at 55°C with Klenow [α-32P]dCTP-labeled XpYp probe in Church mix. Membranes were rinsed three times for 15 min each in Church wash (40 mM sodium phosphate buffer at pH 7.2, 1 mM EDTA, 1% [w/v] SDS) at 55°C and exposed to PhosphorImager screens.
6
Chromatin Conformation Capture Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, control and stimulated cells were fixed in 1% paraformaldehyde for 10 min at room temperature followed by adding glycine (0.6 M, final concentration) to quench the cross-linking reaction. Then cells were suspended in lysis buffer (10 mM Tris-HCl, pH 7.2, 10 mM NaCl, 0.2% NP-40 and protease inhibitor cocktail; Sigma) for 60 min on ice. The nuclei were pelleted, resuspended in nuclear buffer with 0.3% SDS and incubated for 1 hr at 37°C. Triton-X100 (1.8%) was added to sequester the SDS followed by the addition of high concentration BglII and BamHI (NEB) to digest the chromatin overnight at 37°C with gentle shaking. The restriction enzymes were inactivated by the addition of SDS to 1.6% and incubation at 70°C for 15 min. The reaction mixture was diluted with 1x ligase buffer (NEB) and incubated for 1 hr at 37°C. Ligation of DNA was done using T4 ligase for overnight at 16°C. Following reversal of cross-linking and DNA extraction, the samples were subjected to qPCR quantification. The various primer combinations were tested for their amplification efficiency using a control template prepared by mixing mouse rDNA plasmids in equimolar amounts, followed by digestion with BglII and BamHI and subsequent ligation.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!