Plasmid Product Handling Instructions
Plasmid Product Handling Instructions
After receiving your product, please first check the product label to identify the product type. Confirm the appropriate antibiotic resistance, competent-cell strain, and culture temperature for the specific plasmid, and then follow the corresponding instructions below.
I. Ready-to-Use Liquid Plasmid
Product Description
This product is purified from E. coli using our proprietary endotoxin-free plasmid maxi-prep kit. It is a ready-to-use, high-purity, transfection-grade plasmid with a high proportion of the supercoiled form and an extremely low endotoxin level.
It is suitable for a variety of applications, including cell transfection, gene expression, viral packaging, and other molecular biology experiments.
Instructions for Use
1. Upon receipt:
Briefly centrifuge the tube at 3,000–5,000 rpm for 10 seconds to bring the liquid to the bottom of the tube.
2. Usage:
Refer to the plasmid concentration indicated on the product label and calculate the required volume accordingly.
For example, if 1 μg of plasmid DNA is required and the plasmid concentration is 600 ng/μL, use approximately 1.7 μL of the plasmid solution.
3. Storage of the remaining plasmid:
If the entire plasmid preparation is not used at once, aliquot the remaining plasmid and store it frozen to avoid repeated freeze-thaw cycles.
4. Storage Conditions:
-20°C | Shelf Life: 1 year
Precautions
1. For Research Use Only:
This product is intended for research use only and must not be used for clinical applications.
2. Verify the concentration:
Before use, check the actual plasmid concentration indicated on the tube label. The concentration may vary slightly between batches.
3. Dilution:
The plasmid may be diluted to the desired concentration using sterile water or TE buffer. After dilution, use the solution on the same day when possible, or aliquot and store frozen.
4. Aseptic technique:
Maintain strict aseptic conditions when performing cell-related procedures involving the plasmid.
5. Plasmid amplification:
If additional plasmid is required, the plasmid can be transformed into E. coli for amplification and subsequent plasmid preparation, or additional plasmid can be purchased from MiaolingBio.
II. Seed Plasmid Powder
Product Description
This product is supplied as a seed plasmid in lyophilized powder form, with a total amount of approximately 0.5–2 μg.
The plasmid must be transformed into appropriate competent cells, amplified, and purified before use.
The seed plasmid powder should not be used directly for transfection.
Instructions for Use
1. Dissolve the plasmid:
Upon receipt, centrifuge the tube at 5,000 rpm for 1 minute. Then add 20 μL of sterile water to dissolve the plasmid.
2. Transformation:
Thaw the appropriate competent cells on ice. Add 5 μL of the plasmid solution to 100 μL of competent cells and incubate on ice for 30 minutes.
3. Heat shock and recovery:
Heat-shock the cells at 42°C for 60 seconds, followed by incubation on ice for 2 minutes. Add 900 μL of antibiotic-free LB medium and shake at 180 rpm for 45 minutes.
4. Plate the transformed cells:
Centrifuge at 6,000 rpm for 3 minutes. Remove the supernatant, leaving approximately 100 μL, and gently resuspend the cells. Spread the suspension onto an LB agar plate containing the appropriate antibiotic.
5. Incubation and colony assessment:
Incubate the plate upside down overnight under the appropriate culture conditions.
If too many colonies are present, dilute the transformation mixture before plating. If no colonies are observed, repeat the transformation using 10 μL of the plasmid solution.
6. Plasmid amplification:
Pick a single colony and inoculate it into the corresponding antibiotic-containing LB liquid medium. Culture overnight with shaking and perform plasmid preparation according to experimental requirements.
7. Storage Conditions:
-20°C | Shelf Life: 90 days
Precautions
1. Seed plasmid:
This product is supplied as a seed plasmid. Direct transfection efficiency is extremely low. The plasmid must be transformed and amplified before use.
2. Colony number:
Ideally, approximately 10–300 colonies should be obtained on the agar plate. Excessive or insufficient colony numbers may affect subsequent single-colony selection.
3. Sequence verification:
After plasmid preparation, sequence verification is recommended. At least one sequencing reaction should be performed to confirm that the plasmid sequence is correct.
4. Transformation conditions:
Before transformation, verify the plasmid's antibiotic resistance and the appropriate competent-cell strain. Follow the instructions provided for the competent cells.
5. Storage of the remaining plasmid:
After dissolution, store the remaining plasmid solution at -20°C. Repeated freeze-thaw cycles may reduce transformation efficiency. Aliquoting the plasmid before freezing is recommended.
The image above shows an example of typical colony growth on an agar plate.
Both excessive and insufficient colony numbers may affect subsequent plasmid preparation results.
III. Seed Glycerol Stock
This product is a glycerol stock prepared from a single colony after the target plasmid has been transformed into a cloning-competent E. coli strain (e.g., DH5α, Stbl3). The final glycerol concentration is 20%.
The bacterial strain already carries the plasmid you need, so no transformation is required. Simply streak the glycerol stock onto an appropriate agar plate for recovery, then pick a single colony for amplification and plasmid preparation.
Procedure
Upon receipt: Gently shake or flick the glycerol stock tube to ensure that the bacterial suspension is not adhering to the tube wall.
Using a sterile inoculating loop, streak the glycerol stock onto an agar plate containing the appropriate antibiotic using the four-quadrant streaking method.
Incubate the plate inverted at a constant temperature for 12–16 hours.
Always pick a single colony and inoculate it into the appropriate liquid culture medium containing the corresponding antibiotic.
Incubate overnight with shaking. Continue culturing the bacteria as needed for your experiment.
Storage: −80°C
Shelf Life: 90 days
Precautions
Always pick a single colony: Directly inoculating liquid culture from the glycerol stock may result in an impure bacterial population and increase the risk of plasmid mutation or plasmid loss.
Match the antibiotic to the plasmid: Always use the antibiotic corresponding to the plasmid's resistance marker during both agar plate streaking and liquid culture.
Long-term storage: After recovery, fresh bacterial cultures can be used to prepare a new glycerol stock at a final glycerol concentration of 20% and stored at −80°C.
