- Item Description
- Technical Information
- Sample Size: 1 – 4ml bacterial culture
- Expectant Yield: 20-30µg for high-copy plasmids/3-10µg for low-copy plasmid
- Operation Time: 30min or less
- Binding Capacity: up to 30µg
The High Speed Mini Plasmid Kit is designed for rapid isolation of plasmid or cosmid DNA from 1-5ml of bacterial cultures. The modified alkaline lysis method and RNase treatment are used to create cleared cell lysate with minimal genomic DNA and RNA contaminants.
Click on Technical Information or Protocol below for more detailed information.
|Specifications||High-Speed Mini Plasmid Kit|
|Binding Capacity:||30 µg|
|Culture Input:||1-5 ml|
|Culture Type:||Cultured bacterial cells|
|Plasmid Size:||1-15 kb|
|Typical Yield:||10-30 µg|
|Elution Volume:||30-100 µl|
|Operation Time:||<15 minutes|
In the presence of a chaotropic salt, the plasmid DNA in the lysate, binds to the glass-fiber matrix in the spin column. The contaminants are washed off with an ethanol based wash buffer. The purified plasmid DNA is eluted by a low salt elution buffer or water. This procedure does not require DNA phenol extraction or alcohol plasmid. Typical yields are 20-30 µg for high-copy numbered plasmids or 3-10 µg for low-copy numbered plasmids. The purified plasmid DNA is ready to use for restriction enzyme digestion, ligation, PCR, or sequencing reactions. The entire procedure can be completed in under 30 minutes.
The quality of the High-Speed Plasmid Mini Kit is tested on a lot-to-lot basis, by isolating plasmid DNA from a 4 ml overnight E. coli (DH5α) culture, containing plasmid p Bluescript (A600>2U/ml). Following the purification process, a yield of more than 20 µg is expected and the ratio of A260/A280 is between 1.7-1.9. The purified plasmid (1 µg) is used in EcoRI digestion and checked by electrophoresis.
What competitor’s kit is comparable?QIAGENS QIAprep Spin Miniprep Kit QIAGENS QuickLyse Miniprep Kit
Can QIAGEN'S reagents be used with IBI columns?Yes, anything that is “lysis-based” will work.
Is the use of ampicillin or other antibiotics during the cultivation of the E.coli necessary to achieve proper yield with the IBI mini plasmid kit?Yes, in order to achieve good yield, the E.coli must be stressed with an antibiotic.
How do I increase the Yield for this kit?1. Increase your Lysis time 2. Pre-heat the Elution Buffer 3. Perform the Elution step twice 4. With a large/concentrated sample, end-user can add extra PD 1, 2, 3 buffers
What’s the maximum amount of sample for the Mini Plasmid column/tube?The maximum amount of sample for the column/tube is 4 ml.
What is the primary make-up of the Elution buffer?The primary make-up of the buffer is Tris-HCl pH 8.5. This buffer does not contain EDTA. You can also use Pure Water or TE buffer.
Can the plasmid DNA extraction kit be applied on Gram (+) Bacteria?Our plasmid kits are primarily designed to extract plasmid DNA from Gram (-) bacteria such as E. Coli. Gram (+) bacteria has thicker cell walls so the cell lysis buffers provided in the kit do not lyse them readily. However, extraction of plasmid DNA from Gram (+) bacteria can still be achieved with additional treatment. After re-suspending the pelleted bacterial cells in the PD1 Buffer, add lysozyme to give a final concentration of 3 to 5 mg/ml. Incubate the suspension at 37° Celsius for 30-60 minutes (or for a shorter time when 5 mg/ml lysozyme is used.) This treatment weakens the cell wall of the Gram (+) bacteria. Add PD2, and follow the rest of the protocol. For certain Gram (+) bacteria with thin cell walls, such as Lactobacillus, applying a double amount of PD1, PD2, and PD3 Buffer may be enough to lyse the cells. Yet, we still recommend treating Gram (+) bacteria with lysozyme to facilitate cell lysis.
What are the difference and/or purpose of the W1 Buffer vs. the Wash Buffer?W1 contains chaotropic salt and ethanol. The chaotropic salt can inhibit and remove the enzyme activity, such as Taq polymerase and endonuclease. Wash Buffer contains low concentrations of salt and will remove salts and proteins from the final sample.
Was the RNAse A volume increased in the Mini plasmid kits?Yes, the amount of RNAse A was doubled on 9-23-14. The concentration remained the same – 50 mg/ml. The amount of RNAse added to the 25 ml bottle of P D1 is now 100 µl. The amount RNAse added to the 65 ml bottle of P D1 is 260 µl. This is double to what it was. This is for sequencing grade plasmid DNA.
What’s the difference between the High-Speed Mini Plasmid Kit and the I-Blue Mini Plasmid Kits?The differences are 2 X volume of RNAse A and I-Blue Lysis Buffer in the I-Blue Mini. The larger volume of RNAse A increases the yield for larger sample volumes. Hi Speed Mini allows for 1-5 ml and the I-Blue Mini allows for 1-7 ml which will yield more plasmid DNA. All other buffers are the same.
Comparison of Plasmid DNA Extraction Kits
Photo of Gel:Photo 1- Comparision of Percent Recovery
1. IBI brand Purification Kits are equivalent or superior to Brand Q in Yield.
2. IBI brand Purification Kits are equivalent to Brand Q in Purity.