Product Information

Product Description

      This kit uses an improved SDS-alkaline lysis method to lyse cells. The silicon matrix membrane in the centrifugal adsorption column selectively binds to the plasmid DNA in the solution under high salt and low pH conditions, and then removes impurities and other bacterial components through deproteinization solution and rinse solution. Finally, the pure plasmid DNA is eluted from the silicon matrix membrane using a low salt and high pH elution buffer.

Features

1. The silicon matrix membrane in the centrifugal adsorption column is all imported from a world-renowned company. The difference in adsorption amount between columns is very small, and the repeatability is good. This overcomes the disadvantage of unstable membrane quality of domestic test kits.

2. The unique deproteinization liquid formula can effectively remove residual nucleases, even strains rich in nucleases such as JM series and HB101 can be easily removed, effectively preventing plasmids from being degraded by nucleases.

3. Fast and convenient, no toxic reagents such as phenol and chloroform are required, and no ethanol precipitation is required. The obtained plasmid has high yield and good purity, and can be directly used in various molecular biology experiments such as enzyme digestion, transformation, PCR, in vitro transcription, sequencing, etc.

Transportation and storage methods

1) The kit can be stored at room temperature for 12 months without affecting its effectiveness.

2) RNase A is stored in ready-to-use glycerol buffer and shipped at room temperature. Upon receipt, it should be stored at room temperature not exceeding 25°C for at least 6 months, at 4°C for 12 months, and at -20°C for long-term storage.

3) When using for the first time, add all the RNase A in the kit to solution P1 (final concentration 100ug/ml) and store at 2-8℃. If the RNase A in solution P1 is inactivated, there may be trace amounts of RNA remaining in the extracted plasmid. In this case, add more RNase A to solution P1.

4) When the ambient temperature is low, the SDS in solution P2 may become turbid or precipitate. It can be heated in a 37°C water bath for a few minutes to restore clarity. Do not shake violently to avoid excessive foaming.

5) Avoid long-term exposure of reagents to air to prevent volatilization, oxidation, and pH changes. Close the lid of each solution immediately after use.

Precautions

1. All centrifugation steps were performed at room temperature using a conventional benchtop centrifuge capable of reaching 13,000 rpm, such as an Eppendorf 5415C or similar centrifuge.

2. The amount of plasmid extracted is related to factors such as bacterial culture concentration and plasmid copy number. For high-copy plasmids, it is recommended to inoculate a single colony in 1.5-4.5 ml LB medium with appropriate antibiotics and culture overnight for 14-16 hours to extract up to 20µg of pure plasmid. If the extracted plasmid is a low-copy plasmid or a large plasmid larger than 10kb, the amount of bacteria used should be appropriately increased, using 5-10 ml overnight culture, and the amount of P1, P2, and P3 should be increased proportionally. The other steps are the same.

3. The concentration and purity of the obtained plasmid DNA can be tested by agarose gel electrophoresis and UV spectrophotometer. An OD260 value of 1 is equivalent to about 50μg/ml DNA. The electrophoresis may be a single band or two or more DNA bands. This is mainly caused by the different positions of supercoiled plasmids of different degrees, which is related to the length of time the extract is cultivated and the intensity of the extraction operation. Under normal operation, the basic supercoil of our products can exceed 90%.

4. The exact molecular size of plasmid DNA can only be known after linearization by enzyme digestion and comparison with DNA molecular weight marker. Plasmids in a circular or supercoiled state have uncertain migrating positions and their exact size cannot be known by electrophoresis.

5. The elution buffer EB does not contain the chelating agent EDTA, and does not affect downstream enzyme digestion, ligation and other reactions. Water elution can also be used, but the pH should be greater than 7.5. Too low pH affects the elution efficiency. Plasmids eluted with water should be stored at -20°C. If plasmid DNA needs to be stored for a long time, it can be eluted with TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0), but EDTA may affect downstream enzyme digestion reactions, so it can be appropriately diluted when used.

6. This product is for scientific research purposes only!

About the use of balancing solution

1. Introduction: During long-term storage, the nucleic acid adsorption silica gel membrane column will react with the charge/dust in the air and affect its nucleic acid binding ability. After the silica gel column is pretreated with the balance solution, the hydrophobic groups of the silica gel membrane in the column can be greatly reduced, and the nucleic acid binding ability can be improved. Thereby improving the recovery efficiency or yield of the silica gel column. The balance solution is a strong alkaline solution. If you accidentally touch it, please wash it with a large amount of tap water. After use, the bottle cap must be tightly closed to avoid contact with air. Store at room temperature. Precipitation may be generated during storage. Please heat it to 37℃ to make the precipitation completely disappear.

2. Usage: Take a new silica gel membrane adsorption column and place it in a collection tube. Pipette 100μl of the equilibration solution into the column. Centrifuge at 13000 rpm for 1 minute, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube. At this point, the equilibration solution pretreatment column is complete. Continue with the subsequent steps.

How to use

hint:

→ Before using for the first time, please add the specified amount of anhydrous ethanol to the rinse solution WB and protein removal solution PE bottles, mix thoroughly, and after adding, please check the box in time to mark that ethanol has been added to avoid adding it multiple times!

→ Add all RNase A to solution P1, mix well, and store at 2-8℃ after each use.

1. Take 1.5-4.5 ml of overnight cultured bacterial solution, centrifuge at 12,000 rpm for 30 seconds, pour off the supernatant as much as possible, and collect the bacterial cells.

If more than 1.5 ml of bacterial solution is collected, centrifuge and discard the supernatant, then add more bacterial solution to the same 1.5 ml tube and repeat step 1 until enough bacteria are collected.

2. Resuspend the bacterial pellet with 250 μl of solution P1 and vortex until thoroughly suspended.  

If there are bacterial lumps that are not thoroughly mixed, it will affect the lysis, resulting in low extraction volume and purity.

3. Add 250 μl of solution P2, gently invert up and down 6-8 times to fully lyse the bacteria, and leave at room temperature for 4 minutes.

Mix gently, do not shake violently, so as not to shear and break the genomic DNA! The time should not exceed 5 minutes! So as not to damage the plasmid. At this time, the bacterial solution should become clear and viscous. If there are few bacteria, you can proceed to the next step after it becomes clear and viscous quickly. It does not have to be exactly 5 minutes.

4. Add 350 μl of solution P3 and gently invert the tube 6-8 times. When the mixture is fully mixed, a white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 10 minutes and carefully remove the supernatant.

After adding solution P3, mix immediately to avoid local precipitation of SDS.

Equilibration liquid pretreatment adsorption column:

It is a necessary step to use the balance solution to pretreat the silica gel membrane adsorption column. For specific methods, please refer to the previous "About the use of balance solution"

5. Add the supernatant obtained in the previous step to the adsorption column AC (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 30-60 seconds, and discard the waste liquid in the collection tube.

6. Optional step : Add 500 μl of deproteinized PE solution, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

This step is to remove trace nucleases and other impurities. If the strain used is an endA strain such as JM series, HB101 or a wild-type strain, which has a rich nuclease content, this step should be added; if the strain used is a defective strain such as XL-1 Blue, Top10 and DH5α, which has a low nuclease content, this step can be skipped.

7. Add 600μl of washing buffer WB (please check whether anhydrous ethanol has been added first!) , centrifuge at 12,000rpm for 30 seconds, and discard the waste liquid.

8. Add 600 μl of washing buffer WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

9. Place the adsorption column AC back into the empty collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much rinsing solution as possible to prevent residual ethanol in the rinsing solution from inhibiting downstream reactions.

10. Take out the adsorption column AC, put it into a clean centrifuge tube, add 50-100μl elution buffer EB to the middle part of the adsorption membrane (the elution buffer is better if it is heated in a 65-70℃ water bath beforehand), leave it at room temperature for 2 minutes, and centrifuge it at 12,000rpm for 1 minute. If a larger amount of plasmid is needed, add the obtained solution back to the centrifugal adsorption column and centrifuge it for 1 minute.

The larger the elution volume, the higher the elution efficiency. If a higher plasmid concentration is required, the elution volume can be appropriately reduced, but the minimum volume should not be less than 30μl. Too small a volume will reduce the plasmid elution efficiency and plasmid yield.