The Plasmid midiPREP Kit provides a fast, simple, and cost-effective method for the plasmid DNA isolation from the cultured bacterial cells. The Plasmid midiPREP Kit is based on the alkaline lysis of bacterial cells, followed by binding DNA onto the glass fiber matrix of the spin column in the presence of high salt. Phenol extraction and ethanol precipitation are not required, and the high-quality plasmid DNA is eluted in a small volume of the Tris buffer (included in each kit) or water (pH is between 7.0 and 8.5). The plasmid DNA purified with the Plasmid midiPREP Kit is suitable for a variety of routine applications, including the restriction enzyme digestion, sequencing, library screening, in vitro translation, transfection of robust cells, ligation, and transformation. The entire procedure can be completed within 40 minutes.
➤ Ready-to-use DNA for high performance in any downstream application.
➤ Consistent DNA yields from a small amount of the starting material.
➤ Time flexibility.
➤ Quantity of DNA needed.
➤ Molecular weight and size of DNA.
➤ Purity of DNA required.
|Buffer M1||85 ml X 1 bottle||10 ml X 1 bottle|
|Buffer M2||85 ml X 1 bottle||10 ml X 1 bottle|
|Buffer M3||125 ml X 1 bottle||15 ml X 1 bottle|
|Buffer W1||125 ml X 1 bottle, 40 ml X 1 bottle||20 ml X1 bottle|
|Buffer W2 (Add ethanol)||25 ml x 2 bottles (100 ml X 2 bottles)||6 ml X 1 bottle (24 ml X 1 bottle)|
|Buffer BE||50 ml X 1 bottle||5 ml X 1 bottle|
|RNase A (50mg/mL)||200 μl||Has been added into Buffer M1|
|MD Column||20 pieces X 1 bag||2 pieces X 1 bag|
The quality of the Plasmid midiPREP Kit is tested on a lot-to-lot basis to ensure consistent product quality.
➤ Ethanol (96-100%)
➤ Microcentrifuge tubes
➤ Add the provided RNase A solution to the Buffer M1, mix, and store at 2-8°C.
Plasmid midiPREP Kit Protocol
Step 1 Bacterial Cells Harvesting
1. Transfer 50 ml of the bacterial culture to a 50 ml centrifuge tube
2. Centrifuge at 6,000 x g for 5 minute and discard the supernatant.
Step 2 Resuspension
1. Resuspend pelleted bacterial cells in 4 ml of the Buffer M1 (RNase A added)
Step 3 Lysis
1. Add 4 ml of the Buffer M2 and mix thoroughly by inverting the tube 10 times (Do not vortex) and then stand at the room temperature for 2 minutes or until the lysate is homologous.
Step 4 Neutralization
1. Add 6 ml of the Buffer M3 and mix immediately and thoroughly by inverting the tube 10 times (Do not vortex).
2. Centrifuge at 6,000 x g for 10 minutes.
Step 5 Binding
1. Place a MD Column in a 50 ml centrifuge tube.
2. Apply the supernatant (from step 4) to the MD column by decanting or pipetting.
3. Centrifuge at 6,000 x g for 3 minutes.
4. Discard the flow-through and place the MD column back into the same 50 ml centrifuge tube.
Step 6 Wash
1. Add 8 ml of the Buffer W1 into the MD Column.
2. Centrifuge at 6,000 x g for 3 minutes.
3. Discard the flow-through and place the MD column back into the same 50 ml centrifuge tube.
4. Add 12 ml of the Buffer W2 (Ethanol added) into the MD Column.
5. Centrifuge at 6,000 x g for 3 minutes.
6. Discard the flow-through and place the MD column back into the same 50 ml centrifuge tube.
7. Centrifuge at 6,000 x g again for 3 minutes to remove residual Buffer W2.
Step 7 Elution
1. To elute DNA, place the MD column in a new 50 ml centrifuge tube.
2. Add 2 ml of the Buffer BE or water (pH is between 7.0 and 8.5) to the center of each MD column, let it stand for 2 minutes, and centrifuge at 6,000 x g for 3 minutes.
Refer to the table below to troubleshoot problems that you may encounter when you did genomic DNA isolation with the kit.
|Presence of RNA||RNA contamination||Prior to using the Buffer M1, ensure
RNase A is added.
|Plasmid bands was|
smeared on agarose gel
|Plasmid DNA degradation||Keep plasmid preparations on ice or
frozen in order to avoid the plasmid DNA
|Presence of genomic DNA||Genomic DNA contamination||1. Do not overgrow bacterial cultures.
2. Do not incubate more than 5 min after
adding the Buffer M1.
|96-100% ethanol not used||Add ethanol (96-100%) to the
Buffer W2 before use.
|Nuclease contamination||1. Check buffers for nuclease
contamination and replace if necessary.
2. Use the new glass and plastic wares,
and wear the gloves.
|Column overloaded||Decrease the loading volume or lower
the culture density.
|Low yields of DNA||SDS in the Buffer M2|
|The SDS in the Buffer M2 may precipitate
upon storage. If this happens, incubate
the Buffer M2 at 30-40°C for 5 min and
|Incorrect elution conditions||Ensure that the Buffer BE is added into
the center of the MD Column.
|Plasmid lost in the host||Prepare the fresh culture.|
|TE buffer used for DNA elution.||Use the ethanol to precipitate the DNA,
or repurify the DNA fragments and elute
with the nuclease-free water.
|Inhibition of downstream|
|Presence of residual ethanol|
|Following the Wash Step, dry the MD
Column with the additional centrifugation
at 6,000 x g for 3 minutes
|DNA passed through in|
the flow-through or wash
|Inappropriate salt or pH|
conditions in buffers
|Ensure that any buffer prepared in the
laboratory was prepared according to the
|Plasmid DNA floats out of|
wells while running in
|Incomplete removal of the|
|1. Make sure that no residual ethanol
remains in the membrane before
eluting the plasmid DNA.
2. Re-centrifuge or vacuum again if
Related Ordering information
|MB101-0500||Taq DNA polymerase||500 U|
|MB201-0100||Hot Start SUPERMIX||100 Reactions|
|AGT001-0500||AGAROSE Tablet, 0.5g||100 Tablets|
|LD001-1000||Novel Juice Supplied in 6X Loading Buffer||1ml|
➤ Check buffers before use for salt precipitation.
➤ Re-dissolve any precipitate by warming up to 37°C.
➤ Buffers M2, M3 and W1 contain irritants. Wear gloves when handling these buffers.
➤ When using 20 reaction assays, add 100 ml of the ethanol (96-100%) to each bottle of the Buffer W2, and shake before use.
➤ When using 2 reaction assays, add 24 ml of the ethanol (96-100%) to the Buffer W2, and shake before use.
➤ During operation, always wear a lab coat, disposable gloves, and protective equipment.
➤ Research Use Only. Not intended for any animal or human therapeutic or diagnostic uses.