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| Preparation of salt buffers
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| [ 文章来源： | 文章作者：
| 发布时间：2007-08-25|
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Centrifuge the sample through a 0.45 mm filter.
Check the absorbance at 260 nm and, if necessary, dilute the sample down to a concentration that will not exceed the capacity of the column (Dionex NucleoPac™ 9 ´ 250 PA-100; P/N 43011 (anion exchange) column listed bel ow). Typically a 1 ml injection loop is used to inject a 10 mM sample of unlabeled dNTPs Cy-dUTP.
HPLC PurificationColumn used: Dionex NucleoPac™ 9 ´ 250 PA-100; P/N 43011 (anion exchange)
HPLC Equipment: Perkins Elmer Series 200 LC Pump, Perkins Elmer UV/Vis Detector LC 295
1. Equilibrate column in buffer system, 3 mL/min flow rate (general purge method)
1.1. 5 min low salt buffer (LSB) or A
1.2. 2 min gradient to 100% high salt buffer (HSB) or B
1.3. 5 min HSB or B
1.4. 2 min gradient to 60% LSB or A
1.5. 5 min 60% LSB or A
2. Load sample into injection loop. See "Final Considerations" on the next page for sample lot size and other issues.
3. Run gradient (3 mL/min flow, chart recorder 0.5 cm/min)
3.1. 5 min 60% LSB, 40% HSB
3.2. 10 min gradient to 80% HSB
3.3. 1 min gradient to 100% HSB
3.4. 10 min 100% HSB
3.5. 1 min gradient to 60% LSB
3.6. 5 min 60% LSB
Final Preparative Procedures
For a preparative run of 150 nmoles Cy-dUTP (note "Final Considerations"), the recovered fractions of purified Cy-dUTP is eluted in 5 ml of volatile TEAA buffer that can be removed by lyophilization.
Lyophilization and Redilution (Return to HPLC Preparation.)
- Pool fractions of each Cy dye into a 50 mL conical tube. Punch holes into the cap using an 18-gauge needle. Avoid exceeding more than half the volume of the tube, as the samples may have a tendency to splatter during degassing or travel up the tube during lyophilization.
- Freeze the samples on dry ice and transfer them to a lyophilization chamber. Wrap the chamber with foil to protect the samples from light.
- Lyophilize the samples to completion (approx. 12-15 hrs for HPLC-purified samples, 6-10 hrs for Sep-Pak-purified material). HPLC-purified samples will have the appearance of a residue until most of the TEAA is removed.
Note: Steps 4 & 5 are for HPLC-purified samples only.
- Dilute the samples back up to 8-10 mL with 0.22 mm filtered ddH2O and lyophilize until the samples again resemble residue. This time, it may require up to 24 hours of lyophilization.
- Repeat Step 4. Lyophilization is complete when the sample has the appearance of a film and/or specks of solid residue. Resuspend the sample in a small volume of 10 mM phosphate buffer, pH 7.0. Bear in mind that some of the Cy-dUTP will coat the upp er regions of the tube walls and may not be readily visible. Check absorbance of the sample. A560/660/A260 ratios should be around 17 for cy 3 and 25 for cy 5.
Expected overall yields: cy 3: 50-70%; cy 5: 45-65%.
Final Considerations
Currently 150nmol of Cy-dUTP contained in a 10 mM mixture of labeled and unlabeled dNTPs is loaded onto the column listed above, depending on the contents and concentration of the filtrate; PCR flow-through samples tend to be cleaner and may allow for larger injection lots. Thus, the flow-through from PCR and RT reactions may be kept separate so that they can be processed separately. The Sep-Pak purification desalts the sample and is likely to remove other impurities present in the sample. HPLC purif ication can still be performed without Sep-Pak purification, but salt content prohibits injection of more than 50nmol. Furthermore, yields tend to be lower if the sample is not desalted prior to HPLC injection.
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