Plasmid Extraction and Preparation - 上海质因生物科技有限公司

Q: Mini (10–30 μg) is suitable for restriction digestion, sequencing, and PCR verification. Midi (100–200 μg) is suitable for transfection in 6-well plates or 10 cm dishes. Maxi (0.5–1 mg) is suitable for T75 flasks, 15 cm dishes, or small-animal injection. Mega (2–5 mg) is suitable for large-animal studies or repeated transfections, and Giga (10–50 mg) is suitable for large-scale requirements. As a reference, a single well of a 6-well plate usually requires about 2–5 μg, while a T75 flask requires about 10–20 μg.

Mini (10–30 μg) is suitable for restriction digestion, sequencing, and PCR verification. Midi (100–200 μg) is suitable for transfection in 6-well plates or 10 cm dishes. Maxi (0.5–1 mg) is suitable for T75 flasks, 15 cm dishes, or small-animal injection. Mega (2–5 mg) is suitable for large-animal studies or repeated transfections, and Giga (10–50 mg) is suitable for large-scale requirements. As a reference, a single well of a 6-well plate usually requires about 2–5 μg, while a T75 flask requires about 10–20 μg.

Q: A miniprep rapidly purifies plasmid DNA from 1–5 mL of culture with a kit, yielding approximately 10–30 μg in 1–2 business days. Large-scale preparation requires 100–500 mL or more of culture and involves large-scale alkaline lysis, filtration, and chromatographic purification, as well as additional QC testing, so it takes about 3–7 days. A key challenge is preventing mechanical shearing while maintaining a consistent supercoiled fraction.

A miniprep rapidly purifies plasmid DNA from 1–5 mL of culture with a kit, yielding approximately 10–30 μg in 1–2 business days. Large-scale preparation requires 100–500 mL or more of culture and involves large-scale alkaline lysis, filtration, and chromatographic purification, as well as additional QC testing, so it takes about 3–7 days. A key challenge is preventing mechanical shearing while maintaining a consistent supercoiled fraction.

Q: Supercoiled DNA is the natural plasmid conformation in bacteria. It is more compact, crosses cell membranes more readily, resists nuclease degradation inside cells, and supports more efficient transcription, so it generally transfects more efficiently than open-circular or linear DNA. Our transfection-grade plasmids have a supercoiled fraction of ≥90%, verified quantitatively by agarose-gel electrophoresis densitometry.

Supercoiled DNA is the natural plasmid conformation in bacteria. It is more compact, crosses cell membranes more readily, resists nuclease degradation inside cells, and supports more efficient transcription, so it generally transfects more efficiently than open-circular or linear DNA. Our transfection-grade plasmids have a supercoiled fraction of ≥90%, verified quantitatively by agarose-gel electrophoresis densitometry.

Q: Yes. For the same culture volume, low-copy plasmids, such as those derived from pBR322 or pSC101, yield only about one-fifth to one-tenth as much DNA as high-copy plasmids. We improve yield by increasing culture volume, optimizing lysis conditions, and using dedicated purification columns. We recommend choosing one preparation scale larger than you would for a high-copy plasmid, for example Mega instead of Maxi. Turnaround may be extended by 1–2 days.

Yes. For the same culture volume, low-copy plasmids, such as those derived from pBR322 or pSC101, yield only about one-fifth to one-tenth as much DNA as high-copy plasmids. We improve yield by increasing culture volume, optimizing lysis conditions, and using dedicated purification columns. We recommend choosing one preparation scale larger than you would for a high-copy plasmid, for example Mega instead of Maxi. Turnaround may be extended by 1–2 days.

Q: Liquid plasmid DNA can be stored at 4°C for 1–2 weeks or at −20°C or −80°C for long-term storage. Avoid repeated freeze–thaw cycles, which reduce the supercoiled fraction. Lyophilized plasmid DNA is stable for 2–3 years when stored protected from light at −20°C or 4°C and can be reconstituted to the desired concentration in sterile water or TE buffer (pH 8.0). Glycerol stocks can be stored at −80°C for more than five years and streaked for recovery when needed. Large-scale plasmid preparations should be divided into multiple aliquots for storage.

Liquid plasmid DNA can be stored at 4°C for 1–2 weeks or at −20°C or −80°C for long-term storage. Avoid repeated freeze–thaw cycles, which reduce the supercoiled fraction. Lyophilized plasmid DNA is stable for 2–3 years when stored protected from light at −20°C or 4°C and can be reconstituted to the desired concentration in sterile water or TE buffer (pH 8.0). Glycerol stocks can be stored at −80°C for more than five years and streaked for recovery when needed. Large-scale plasmid preparations should be divided into multiple aliquots for storage.

What is the difference between research-grade and transfection-grade plasmids, and which should I choose?

<p>Research-grade plasmids are not subjected to endotoxin removal and have a supercoiled fraction of ≥80%. They are suitable for in vitro molecular-biology applications such as restriction digestion, sequencing, PCR, and subcloning and are more economical. Transfection-grade plasmids are purified to an endotoxin level of <0.05 EU/μg and have a supercoiled fraction of ≥90%. They are suitable for cell transfection, microinjection, and in vitro transcription. Choose transfection grade whenever the downstream application involves cells or animals.</p>

What is endotoxin, and why should endotoxin-free plasmids be used for transfection?

Endotoxin, or lipopolysaccharide (LPS), is a component of the E. coli outer membrane. It is released during cell lysis and can co-purify with plasmid DNA. Endotoxin competes with plasmid DNA for binding to the cell membrane and reduces transfection efficiency, activates immune cells through TLR4 to release inflammatory factors, interferes with target-gene expression, and may cause systemic inflammation after in vivo injection. Depending on cell sensitivity, endotoxin specifications of <0.05, <0.01, or <0.005 EU/μg can be selected.

How should I choose among Mini, Midi, Maxi, Mega, and Giga plasmid preparations?

Mini (10–30 μg) is suitable for restriction digestion, sequencing, and PCR verification. Midi (100–200 μg) is suitable for transfection in 6-well plates or 10 cm dishes. Maxi (0.5–1 mg) is suitable for T75 flasks, 15 cm dishes, or small-animal injection. Mega (2–5 mg) is suitable for large-animal studies or repeated transfections, and Giga (10–50 mg) is suitable for large-scale requirements. As a reference, a single well of a 6-well plate usually requires about 2–5 μg, while a T75 flask requires about 10–20 μg.

What is the difference between plasmid miniprep and large-scale preparation, and why does large-scale preparation take longer?

A miniprep rapidly purifies plasmid DNA from 1–5 mL of culture with a kit, yielding approximately 10–30 μg in 1–2 business days. Large-scale preparation requires 100–500 mL or more of culture and involves large-scale alkaline lysis, filtration, and chromatographic purification, as well as additional QC testing, so it takes about 3–7 days. A key challenge is preventing mechanical shearing while maintaining a consistent supercoiled fraction.

Why is the supercoiled fraction important?

Supercoiled DNA is the natural plasmid conformation in bacteria. It is more compact, crosses cell membranes more readily, resists nuclease degradation inside cells, and supports more efficient transcription, so it generally transfects more efficiently than open-circular or linear DNA. Our transfection-grade plasmids have a supercoiled fraction of ≥90%, verified quantitatively by agarose-gel electrophoresis densitometry.

Can low-copy plasmids be prepared, and what yield can be expected?

Yes. For the same culture volume, low-copy plasmids, such as those derived from pBR322 or pSC101, yield only about one-fifth to one-tenth as much DNA as high-copy plasmids. We improve yield by increasing culture volume, optimizing lysis conditions, and using dedicated purification columns. We recommend choosing one preparation scale larger than you would for a high-copy plasmid, for example Mega instead of Maxi. Turnaround may be extended by 1–2 days.

How should a plasmid be stored after delivery, and how long is it stable?

Liquid plasmid DNA can be stored at 4°C for 1–2 weeks or at −20°C or −80°C for long-term storage. Avoid repeated freeze–thaw cycles, which reduce the supercoiled fraction. Lyophilized plasmid DNA is stable for 2–3 years when stored protected from light at −20°C or 4°C and can be reconstituted to the desired concentration in sterile water or TE buffer (pH 8.0). Glycerol stocks can be stored at −80°C for more than five years and streaked for recovery when needed. Large-scale plasmid preparations should be divided into multiple aliquots for storage.