T4 dna ligase reaction buffer

Penicillin-Streptomycin (P0781), PMA (P8139), βME (M3148), Hank’s balanced salt solution (H9269), DPBS (D8537) and polybrene (107689) were purchased from Sigma Aldrich. DMEM (BE12-604F, Lonza), Trypsin/EDTA (BE17-161E, Biowhittaker), FBS (10270–106, GIBCO) and LPS-EK Ultrapure from the E. coli K12 strain (tlrl-peklps) from Invivogen. Opti-MEM I Reduced Serum Medium (11058021) from ThermoFisher Scientific and Puromycin (540222) from VWR. BsmB1 restriction enzyme (R0580), T4 DNA ligase (M0202) and T4 DNA ligase reaction buffer (B0202) were from New England Biolabs. PureYield Plasmid Miniprep System (A1222) was from Promega.

ChIP DNA was end repaired by incubation at 20°C for 30 minutes with 3 U of T4 DNA Polymerase (NEB, Hitchin, UK), 10 U of Polynucleotide Kinase (NEB), 2 U DNA Polymerase I Large (Klenow) fragment (NEB), 1× T4 DNA ligase reaction buffer (NEB) and 400 nM dNTPs. The enzymes were then heat inactivated at 75°C for 20 minutes, after which the DNA was ethanol precipitated. A tail of 'A' bases was added to the 3’ ends of the DNA by incubation with 5 U Klenow Fragment (3’-5’ exo-; NEB), 200 nM dATP and 1X buffer 2 (NEB) at 37°C for 30 minutes. The enzymes were heat inactivated and cleaned up as before. Barcoded Ilumina paired end adaptors were then ligated to the processed ChIP DNA by incubation with 300 U of T4 DNA ligase (NEB), 1× T4 DNA ligase buffer (NEB), 7.5% PEG-6000 and 2 pmol of annealed Illumina adaptors for 3 h at room temperature. Ligated DNA was purified using MinElute PCR columns (Qiagen, Hilden, Germany) and eluted in 10 μl water.

To prepare DNA substrates for microscopy, 125 μg of λ-phage DNA was mixed with two oligos (2 μM oligo Lab07 (/5Phos/AGG TCG CCG CCC/3BioTEG) and 2 μM oligo Lab06 (/5Phos/GGG CGG CGA CCT/3BioTEG) in 1× T4 DNA ligase reaction buffer (NEB B0202S) and heated to 70°C for 15 min followed by gradual cooling to 15°C for 2 hours. One oligo will be annealed with the overhand located at the left cohesive end of DNA, and the other oligo will be annealed with the overhang at the right cohesive end. After the oligomer hybridization, 2 μL of T4 DNA ligase (NEB M0202S) was added to the mixture and incubated overnight at room temperature to seal nicks on DNA. The ligase was inactivated with 2 M NaCl, and the reaction was resolved over a custom-packed S-1000 gel filtration column (GE) to remove excess oligonucleotides and proteins.
For the preparation of 60 bp short DNA labeled with ATTO647N, Oligo1 (/5BioTEG/ACG AAG TCT TAT GGC AAA ACC GAT GGA CTA TGT TTC GGG TAG CAC CAG AAG TCT ATA ACA) and ATTO647N-tagged Oligo2 (5TGT TAT AGA CTT CTG GTG CTA CCC GAA ACA TAG TCC ATC GGT TTT GCC ATA AGA CTT CGT /3ATTO647N/) were purchased from IDT and annealed by combining 20 μM Oligo1 with 20 μM Oligo2. The annealing process involved heating to 75°C for 10 minutes, followed by a gradual cooling to 22°C over a period of 1 hour in a thermal cycler.

To prepare DNA substates for single-molecule imaging, 125 μg of λ-phage DNA was mixed with two oligos (2 μM oligo Lab07 and 2 μM oligo Lab09) in 1× T4 DNA ligase reaction buffer (NEB B0202S) and heated to 70 °C for 15 min followed by gradual cooling to 15 °C for 2 h. One oligo will be annealed with the overhand located at the left cohesive end of DNA, and the other oligo will be annealed with the overhand at right cohesive end. After the oligomer hybridization, 2 μl of T4 DNA ligase (NEB M0202S) was added to the mixture and incubated overnight at room temperature to seal nicks on DNA. The ligase was inactivated with 2 M NaCl, and the reaction was injected to an S-1000 gel filtration column (GE) to remove excess oligonucleotides and proteins.

For total vtRNA measurements, 50 μg of total RNA was treated with calf intestinal phosphatase (CIP) (NEB) for 1 h at 37 °C according to manufactures recommendation. RNA was extracted with phenol:chloroform:isoamyl alcohol [25:24:1 (vol/vol)] followed by ethanol precipitation. A 5 μg of CIP-treated RNA was phosphorylated with ATP and T4 polynucleotide kinase prior to a biospin 6 (BioRad) clean up. Ligations were performed similar to as previously described^{59 (link)}. For ligation, all reactions consist of 100fmol bridge oligonucleotide, 200fmol radiolabeled ligation oligonucleotide, 5 μg RNA, 8% PEG 8000, 1× T4 DNA ligase reaction buffer (NEB), and 10 units T4 DNA ligase (NEB). Before adding T4 DNA ligase, the reaction mixture was denatured at 95 °C for 1 min, cooled to 65 °C for 5 min, and 37 °C for 10 min, the ligase was added to the reaction mixture and incubated at 30 °C for 4 h. Reactions were terminated by heat inactivation at 75 °C for 10 min and subsequently separated using denaturing 8 M urea 10% polyacrylamide gels and imaged using a PhosphorImager.