HSB, 800 mL
LSB, 1000 mL
*Filter, and then sparge both buffers before use. Makes enough for approx. 9 runs & 2 purges.
Sample Preparation, Loading, and Elution:
Immediately wash (pre-load protocol) and process any subsequent lots, reusing the column only twice for each lot. If the column is allowed to sit for more than 5 minutes, yields may decrease 5-10% or more. Avoid reusing the column more than two times (a total of three uses), as yields may begin to diminish. Pool the lots together, check UV absorbance, and calculate yield. Expected yields for this stage are 70-80% for either Cy dye.
1. Equilibrate column in buffer system, 3 mL/min flow rate (general purge method)
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)
Lyophilization and Redilution(Return to HPLC Preparation.)
Note: Steps 4 & 5 are for HPLC-purified samples only.
Expected overall yields: cy 3: 50-70%; cy 5: 45-65%.
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.