Buffer YPI, YPII, YPIII, SE, KB, DNA Wash Buffer, Glass beads, Lyticase solution, RNase A, Elution Buffer, ezBindTM DNA Mini Columns(50), User Manual(1).
Introduction
EZgeneTM yeast plasmid kit is designed for rapid and reliable isolation of high-quality plasmid DNA from yeast cultures. Utilizing the reversible nucleic acid-binding properties of our matrix, the plasmid DNA is bound to the matrix while proteins and other unwanted impurities are eliminated by wash buffer. Pure DNA is then eluted. Purified DNA can be directly used in downstream applications such as PCR, restriction digestion, and Southern Blot.
The Yeast Plasmid Mini Kit combines the power of spin column technology with the lyticase, glass beads and alkaline-SDS lysis of yeast cells to yield high quality plasmid DNA in less than 1 hour. The mini spin columns facilitate the binding, washing, and elution steps, thus enabling multiple samples to be processed simultaneously. The actual plasmid yields depend on copy numbers, yeast strain, and conditions of growth. Because of low copy numbers, the maximum yield from 5 mL yeast culture is around 1 µg.
This protocol has been successfully used to isolate autonomous plasmids from S. cerevisiae. As a modified alkaline lysis procedure, genomic DNA is virtually eliminated from the preparation. Note that all centrifugation steps should be carried out at room temperature.
Storage and Stability
All EZgeneTM Yeast Plasmid Mini Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows: Buffer YP I/RNase A mix at 4oC, Lyticase solution at -20oC, all other components at room temperature.
Capacity
The binding capacity for the mini column is 40 µg of plasmid DNA.
Kit Components
|
Catalog# |
YD1271-00 |
YD1271-01 |
YD1271-02 |
|
Preps |
4 |
50 |
250 |
|
ezBindTM DNA Mini Columns |
4 |
50 |
250 |
|
Buffer YPI |
1.5 mL |
15 mL |
70 mL |
|
Buffer YPII |
1.5 mL |
15 mL |
70 mL |
|
Buffer YPIII |
2 mL |
20 mL |
100 mL |
|
Buffer SE |
3.0 mL |
30 mL |
135 mL |
|
Buffer KB |
3.0 mL |
28 mL |
135 mL |
|
DNA Wash Buffer |
2 mL |
15 mL |
3 x 24 mL |
|
Glass beads |
250 mg |
2.7 g |
13 g |
|
Lyticase solution |
150 µL |
2 mL |
8 mL |
|
RNase A |
10 µL |
130 µL |
650 µL |
|
Elution Buffer |
600 µL |
10 mL |
30 mL |
|
Manual |
1 |
1 |
1 |
Before Starting
Briefly examine this booklet and become familiar with each step. Prepare all components and have the necessary materials ready before starting.
- Dilute DNA Wash Buffer with absolute ethanol as follows：
YD1271-00: Add 8 mL absolute ethanol
YD1271-01: Add 60 mL absolute ethanol
YD1271-02: Add 96 mL absolute ethanol per bottle
- Add vial of RNase A to bottle of YPI and store at 4oC
Materials to Be Provided by Users
- Tabletop micro-centrifuge and nuclease-free 1.5 mL tubes.
- Water bath set to 30 oC.
- Absolute ethanol (96%-100%) - Do not use other alcohols.
- ß-mercaptoethanol
Trouble Shooting Guide
|
Problems |
Possible Reasons |
Suggested Improvements |
|
Low DNA yield |
Poor cell lysis |
Do not use more than 5 mL (with high copy plasmids or 10 mL with low copy plasmids) culture with the basic protocol. |
|
Cells may not be dispersed adequately |
Completely disperse the cell suspension by vortexing after adding Buffer YPI. After adding Buffer YPII, mix completely to obtain a clear lysate. |
|
|
Buffer YP II, if not tightly closed, may need to be replaced. |
Prepare as follows: 0.2 N NaOH, 1% SDS. |
|
|
Yeast culture overgrown or not fresh.
|
Do not incubate cultures for more than 24 hr at 30oC. Storage of cultures for extended periods prior to plasmid isolation is detrimental. |
|
|
Low copy number plasmid used
|
Increase culture volume to 10 mL and scale up buffer volume. |
|
|
No DNA eluted
|
Extended centrifugation during elution step at higher than 13,000 x g. Matrix may be present in eluate and cause abnormal OD readings. |
If the centrifugation speeds higher than specified, some matrix residues may be co-purified with the plasmid DNA, but it will not interfere with PCR or restriction digests. Centrifuge the samples at suggested speed. |
|
Incomplete mixing with Buffer YPI |
Repeat the procedure, this time making sure to vortex the sample with Buffer YPIII immediately and completely. |
|
|
Insufficient mixing with Buffer YPII |
Increase incubation time with Buffer YPII. Ensure that no visible cell clumps remain. |
|
|
No DNA eluted |
DNA Wash Buffer not diluted with absolute ethanol. |
Prepare DNA Wash Buffer as instructed above. |
|
High molecular weight DNA contamination of product |
Over mixing of cell lysate upon addition of Buffer YPII.
|
Do not vortex or mix aggressively after adding Buffer YPII. Adequate mixing is obtained by simply inverting and rotating tube to cover walls with viscous lysate. |
|
Optical densities do not agree with DNA yield on agarose gel |
Trace contaminants eluted from column increase A 260. |
Make sure to wash column as instructed. Alternatively, rely on agarose gel/ethidium bromide electrophoresis for quantization. |
|
RNA visible on agarose gel |
RNase A not added to Buffer YPI. |
Add RNase A to Buffer YPI. |
|
Plasmid DNA floats out of well while loading agarose gel
|
Ethanol not completely removed from column following wash steps. |
Centrifuge column as instructed. |
Operating Protocol
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