The air-dried bead-nuclei mixture was resuspended in 100 μL 1× T4 DNA ligase Buffer with ATP containing 1 U/μL T4 Polynucleotide Kinase (PNK) (Thermo), and incubated at 37 °C for 1 h to phosphorylate ligated bridge adaptors. Following incubation, 90 μL 10× T4 DNA ligase Buffer with ATP, 6 μL T4 DNA ligase (5 U/μL; Thermo), and 804 μL of H2O were added to the reaction mix. In situ proximity ligation was then carried out at room temperature for 4 h.
T4 dna ligase buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
T4 DNA ligase buffer is a buffer solution designed to be used in conjunction with T4 DNA ligase, an enzyme that catalyzes the formation of phosphodiester bonds between adjacent 3'-hydroxyl and 5'-phosphate termini in double-stranded DNA molecules. The buffer provides the optimal ionic conditions and pH for the enzymatic activity of T4 DNA ligase.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (15 mentions)
Transt1 e coli, by Transgene (2 mentions)
Bovine serum albumin (bsa), by New England Biolabs (2 mentions)
Fastdigest lgui sapi, by Thermo Fisher Scientific (2 mentions)
Fd buffer, by Thermo Fisher Scientific (2 mentions)
T4 polynucleotide kinase pnk, by Thermo Fisher Scientific (1 mentions)
Pcr purification kit, by Qiagen (1 mentions)
Precision plus protein kaleidoscope prestained protein standard, by Bio-Rad (1 mentions)
Quick ligation kit, by New England Biolabs (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Calf intestinal phosphatase, by New England Biolabs (1 mentions)
Custom oligonucleotides, by Integrated DNA Technologies (1 mentions)
Pcr pure kit, by Magen Biotechnology Co (1 mentions)
Bsai hf, by New England Biolabs (1 mentions)
Gel pure kit, by Magen Biotechnology Co (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Biotin 14 datp, by Thermo Fisher Scientific (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Instantblue protein stain, by Abcam (1 mentions)
Bsai restriction enzyme, by New England Biolabs (1 mentions)
18 protocols using t4 dna ligase buffer
1
Phosphorylation and Proximity Ligation
2
Engineered Silk-Elastin-Like Proteins
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pET24a(+)
was previously modified for PRe-RDL
(recursive directional ligation by plasmid reconstruction) cloning.21 (link) PRe-RDL vectors containing the genes encoding
ESm = (GSGVP)m (with m = 40, 60, and 80) were developed
previously.35 (link) Custom oligonucleotides for
the construction of the silk blocks SnQ = (GAGAGAGQ)n were synthesized
by Integrated DNA Technologies Inc. Restriction enzymes, calf-intestinal
phosphatase (CIP), and Quick Ligation kit were ordered from New England
Biolabs, and T4 DNA ligase buffer was purchased from Invitrogen. The
DNA miniprep, gel purification, and PCR purification kits were purchased
from Qiagen Inc. Chemically competent Escherichia coli cells (EB5Alpha and BL21 (DE3)) were purchased from EdgeBioSystems,
and the TBdry growth media was purchased from MO BIO Laboratories
Inc. The 4–20% Ready Gel Tris–HCl precast gels and Precision
Plus Protein Kaleidoscope Prestained Protein Standard were purchased
from BioRad.
was previously modified for PRe-RDL
(recursive directional ligation by plasmid reconstruction) cloning.21 (link) PRe-RDL vectors containing the genes encoding
ESm = (GSGVP)m (with m = 40, 60, and 80) were developed
previously.35 (link) Custom oligonucleotides for
the construction of the silk blocks SnQ = (GAGAGAGQ)n were synthesized
by Integrated DNA Technologies Inc. Restriction enzymes, calf-intestinal
phosphatase (CIP), and Quick Ligation kit were ordered from New England
Biolabs, and T4 DNA ligase buffer was purchased from Invitrogen. The
DNA miniprep, gel purification, and PCR purification kits were purchased
from Qiagen Inc. Chemically competent Escherichia coli cells (EB5Alpha and BL21 (DE3)) were purchased from EdgeBioSystems,
and the TBdry growth media was purchased from MO BIO Laboratories
Inc. The 4–20% Ready Gel Tris–HCl precast gels and Precision
Plus Protein Kaleidoscope Prestained Protein Standard were purchased
from BioRad.
3
Tet Strand Circularization Protocol
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The non-circularized tet strand was synthesized with a 5′ phosphate and column purified by PAN facilities, Stanford University. 700 pmol non-circularized tet was combined with 1.4 μmol tet-join in 1.4 mL T4 DNA ligase buffer (Invitrogen), and the solution was incubated at 90 C for 5 min, then left at RT for 10 min, to allow tet-join to form a splint connecting the 3′ and 5′ ends of tet. 50 U (Weiss units) of T4 DNA ligase (Invitrogen) was then added, and the reaction was incubated at RT for 3 hours. 200 U of ExoI (NEB) and 200 U of ExoIII (NEB) were then added, and the solution was incubated overnight at 37 C, phenol-chloroform extracted, ethanol precipitated, and resuspended in water. Samples were then gel purified using 8 % denaturing PAGE gels containing 8M Urea and 1X TBE. Bands were visualized by UV shadowing, cut, and eluted overnight at RT in buffer containing 500 mM ammonium acetate, 10 mM magnesium acetate, and 2 mM EDTA. The samples were then ethanol precipitated, dried down, and reconstituted in water.
4
Tet Strand Circularization Protocol
5
One-step Multiplex CRISPR Cloning
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Multiple sgRNA expression cassettes mixtures (1−2 µg) in an equimolar ratio were digested in a 50-μl reaction with 1 µl of Bsa I-HF (NEB, USA). Digested products were purified using a PCR Pure kit (Magen, China).
Ligation reactions (10 μl) were set up with 1 × T4 DNA ligase buffer, 5 U of T4 DNA ligase (Thermo, USA), 50 ng of Cas9VL, and 20 molar ratios of each digested sgRNA expression cassette (mixture). Reactions were incubated at 22 °C for 1 h. For two to four sgRNA expression cassettes, 10 µl of the ligation mixture was transformed into 50 µl of chemically competent transT1 E. coli (Transgene, China). In cases with five or more sgRNA expression cassettes, 100 µl of chemically competent transT1 E. coli are required. A schematic diagram of the assembly of multiple sgRNA expression cassettes is shown in Fig.2d .
We prepared eight pairs of Bsa I-site primers; therefore, eight different sgRNA expression cassettes can be assembled by one-step Golden Gate assembly that is compatible with the BioBrick Standard Assembly. Biobricks of four sgRNA expression cassettes can be amplified using Bb-F and Bb-R primers from four ligation products of sgRNA expression cassettes and assembled into Biobricks site of pHNCas9. This strategy is suitable for ligating more than eight sgRNA expression cassettes into pHNCas9. Bb-F and Bb-R primer are provided in TableS2 .
Ligation reactions (10 μl) were set up with 1 × T4 DNA ligase buffer, 5 U of T4 DNA ligase (Thermo, USA), 50 ng of Cas9VL, and 20 molar ratios of each digested sgRNA expression cassette (mixture). Reactions were incubated at 22 °C for 1 h. For two to four sgRNA expression cassettes, 10 µl of the ligation mixture was transformed into 50 µl of chemically competent transT1 E. coli (Transgene, China). In cases with five or more sgRNA expression cassettes, 100 µl of chemically competent transT1 E. coli are required. A schematic diagram of the assembly of multiple sgRNA expression cassettes is shown in Fig.
We prepared eight pairs of Bsa I-site primers; therefore, eight different sgRNA expression cassettes can be assembled by one-step Golden Gate assembly that is compatible with the BioBrick Standard Assembly. Biobricks of four sgRNA expression cassettes can be amplified using Bb-F and Bb-R primers from four ligation products of sgRNA expression cassettes and assembled into Biobricks site of pHNCas9. This strategy is suitable for ligating more than eight sgRNA expression cassettes into pHNCas9. Bb-F and Bb-R primer are provided in Table
6
DNA Fragment Ligation Protocol
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The resulting DNA fragments were then ligated by adding a master mix containing nuclease-free water (657 μL for the 5 M to 100 k samples or 328.5 μL for the 50 k to 1 k samples), 10× T4 DNA ligase buffer (120 μL for the 5 M to 100 k samples or 60 μL for the 50 k to 1 k samples; Thermo Fisher Scientific), 10% Triton X-100 (100 μL for the 5 M to 100 k samples or 50 μL for the 50 k to 1 k samples), 20 mg/mL BSA (12 μL for the 5 M to 100 k samples or 6 μL for the 50 k to 1 k samples; New England Biolabs) and 5 Weiss U/μL T4 DNA ligase (5 μL for the 5 M to 100 k samples or 3.5 μL for the 50 k to 1 k samples in two instalments; Thermo Fisher Scientific). Samples were mixed by inversion and incubated at 20 ° C with gentle rotation (20 rpm) for 4 h.
7
Construction of CRISPR Vector with AtMYB75
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PEASY–AtMYB75 was digested with Kpn I and Spe I restriction enzyme (Thermo, USA). BioBrick OEAtMYB75 fragments were separated by agarose gel electrophoresis and purified using gel Pure kit (Magen, China). pHNCas9 was digested by Kpn I and Xba I restriction enzyme (Thermo, USA) using PCR pure kit. Ligation reactions (10 μl) were set up with 1 × T4 DNA ligase buffer, 1 U of T4 DNA ligase (Thermo, USA), 50 ng of pHNCas9, and five molar ratios of BioBrick OEAtMYB75. Reactions were incubated at 22 °C for 1 h. A total of 5 µl of the ligation mixture was transformed into 25 µl of chemically competent transT1 E. coli (Transgene, China). The new CRISPR vector containing BioBrick OEAtMYB75 was named pHNCas9:OEAtMYB75 (Fig. 7a ).
8
Bacterial Transformation Protocol
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Five microliters of the prepared insert was ligated with 1 μl of cleaved expression vector (∼100 ng/μl) in 11.8 μl water, 2 μl T4 DNA ligase buffer (10×) and 0.2 μl T4 DNA ligase (5 U/μl, Thermo Fischer Scientific Inc., Waltham, MA, USA) at room temperature for 2 h. Ligation reaction was transferred to 150 μl of ice cold E. coli Rosetta(DE3) and was incubated for 30 min on ice. Cells were heat shocked at 41°C for 5 min if using deep well plate or 40°C for 4.5 min if using microcentrifuge tube. After 5 min of incubation on ice, 700 μl of pre-warmed TSB + Y was added before incubation at 37°C for 30–60 min, gentle mixing. Samples were centrifuged for 2 min at 700 RCF, ∼620 μl of media was removed and cells were re-suspended before plating on pre-warmed agar plates.
9
Biotin Labeling and Proximity Ligation of DNA-Dendrimer Complexes
10
SUMO Protein Purification Protocol
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RB19 was supplied from Acros Organics (Pittsburgh, PA, USA). BsaI restriction enzyme was supplied from New England Biolabs (Ipswich, MA, USA). T4 DNA ligase and T4 DNA ligase buffer were supplied from Thermofisher Scientific (Waltham, MA, USA). All media components and ampicillin antibiotic were from Fischer Scientific chemicals (Pittsburgh, PA, USA). SUMO protease enzyme was provided by GECCO (Groningen, The Netherlands). InstantBlueTM protein stain was purchased from Expedeon (San Diego, CA, USA). The QiaPrep Spin Miniprep kit was purchased from Qiagen (Hilden, Germany).
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