PCR inserts were amplified using Phusion High‐Fidelity DNA polymerase (M0530L, NEB). Amplification primers were designed to append a 30 base pair overlap with the linear ends of restriction‐digested recipient vectors. Linearized vector backbones were dephosphorylated by calf intestinal phosphatase (M0290S, NEB). All inserts and vectors were purified from a 0.9% agarose gel prior to isothermal assembly (D4002, Zymo Research). 50 ng of linearized vector DNA was combined with isomolar amounts of purified insert(s). 2.5 μl DNA mix was incubated with 7.5 μl isothermal assembly master mix at 50°C for 20 min. Product of the isothermal assembly reaction was transformed into NEB Stable cells (C3040H, NEB). Transformed cells were plated on plates of LB media (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl) containing 1.5% agar. 100 µg/ml ampicillin or 50 µg/ml kanamycin were included in bacterial cultures, where appropriate. All cultures and plates were grown overnight at 34°C. Overnight cultures were pelleted at 3,000 g for 10 min and plasmid DNA was purified using a Qiagen miniprep kit (27106, Qiagen). Sequences were verified by Sanger sequencing (Eton Bioscience Inc).
D4002
Manufactured by Zymo Research
The D4002 is a laboratory equipment product manufactured by Zymo Research. It is a device designed for the efficient extraction and purification of DNA from various biological samples. The core function of the D4002 is to facilitate the isolation and retrieval of DNA for further analysis and applications.
Automatically generated - may contain errors
5 protocols using d4002
1
Isothermal Assembly of DNA Constructs
2
Isothermal Assembly Cloning Protocol
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PCR fragments were generated using 2X phusion master mix (M0531S, NEB, Ipswich, MA) and insert-specific primers that appended a 30 bp overlap with target DNA. Vector backbones were linearized by restriction enzyme and dephosphorylated by calf intestinal phosphatase (M0290S, NEB, Ipswich, MA). Prior to assembly, all DNA fragments were gel purified (D4002, Zymo Research, Irvine, CA). Linearized vector DNA (50 ng) was combined with isomolar amounts of purified insert(s); 5 ul of the resulting DNA mix was added to isothermal assembly master mix and incubated at 50°C for 20 min [68 (link)]. Assembled product was transformed into NEB Stable competent cells (C3040H, NEB, Ipswich, MA) and plated on LB + agar plates (plus appropriate antibiotics) to isolate single isolates. Single isolates were grown in LB broth + antibiotics, and plasmid DNA was purified using a Qiagen miniprep kit (#27106, Qiagen, Hilden, Germany). Sequences were verified by Sanger sequencing (Eton Bioscience, San Diego, CA).
3
Isothermal Assembly of Plasmid Constructs
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PCR inserts were amplified with Phusion High-Fidelity DNA Polymerase (M0530L, NEB). Amplification primers were designed with a 30 bp overlap with the linear ends of restriction-digested vectors. Linearized vectors were dephosphorylated using calf intestinal phosphatase (M0290S, NEB). All inserts and vectors were purified using 0.9% agarose gel prior to isothermal assembly (D4002, Zymo Research). 50 ng of linearized vector DNA was mixed with an isomolar amount of purified insert(s). 2.5 μl of DNA mix was incubated with 7.5 μl isothermal assembly master mix at 50°C for 20 minutes. Product of the isothermal assembly reaction was transformed into NEB Stable cells (C3040H, NEB). Transformed cells were plated on 1.5% agar plates with LB media (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl). All cultures and plates were grown at 34°C overnight and included 100 μg/ml ampicillin. Overnight cultures were pelleted at 3,000 g for 10 min and plasmid DNA was extracted and purified using a Qiagen miniprep kit (27106, Qiagen). Sequences were verified by Sanger sequencing (Eton Bioscience Inc).
4
Isothermal Assembly of Recombinant Plasmids
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PCR inserts were amplified using Phusion High-Fidelity DNA polymerase (M0530L, NEB). Amplification primers were designed with a 30 base pair overlap with the linear ends of restriction-digested recipient vectors. Linearized vector backbones were dephosphorylated by calf intestinal phosphatase (M0290S, NEB). All inserts and vectors were purified from a 0.9% agarose gel prior to isothermal assembly (D4002, Zymo Research). 50ng of linearized vector DNA was combined with isomolar amounts of purified insert(s). 2.5µL DNA mix was incubated with 7.5ll isothermal assembly master mix at 50°C for 20 min. Product of the isothermal assembly reaction was transformed into NEB Stable cells (C3040H, NEB). Transformed cells were plated on plates of LB media (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) containing 1.5% agar, 100µg/mL ampicillin or 50µg/mL kanamycin were included in bacterial cultures, where appropriate. All cultures and plates were grown overnight at 34°C. Overnight cultures were pelleted at 3,000g for 10 min and plasmid DNA was purified using a Qiagen miniprep kit (27106, Qiagen). Sequences were verified by Sanger sequencing (Eton Bioscience Inc).
5
Isothermal Assembly of DNA Constructs
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PCR products were amplified using Phusion High-Fidelity DNA polymerase (M0530L, NEB). Amplification primers were designed with a 30-base pair overlap with the recipient vectors. Linearized vector backbones were dephosphorylated by calf intestinal phosphatase (M0290S, NEB). All inserts and vectors were purified from a 1% agarose gel prior to isothermal assembly (D4002, Zymo Research). In total, 20 ng of linearized vector DNA was combined with isomolar amounts of purified insert(s). Overall, 2.5 µL DNA mix was incubated with 7.5 µL isothermal assembly master mix at 50 °C for 20 min. The product of the isothermal assembly reaction was transformed into NEB Stable cells (C3040H, NEB). Transformed cells were plated on plates of LB media (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) containing 1.5% agar, 100 μg/mL ampicillin or 50 μg/mL kanamycin were included in bacterial cultures. All plates were grown overnight at 34 °C and transformed colonies were grown overnight at 34 °C. Overnight cultures were pelleted at 3000 × g for 10 min and plasmid DNA was purified using a Qiagen miniprep kit (27106, Qiagen). Sequences were verified by Sanger sequencing (Eton Bioscience Inc).
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