Preparation of salt buffers
The buffers are used for both HPLC purification and Sep-Pak purification.
High salt buffer (HSB):
- 1M triethylammonium acetate, pH 8.0
- 10% acetonitrile
Low salt buffer (LSB):
- 25mM triethylammonium acetate, pH 8.0
- 10% acetonitrile (this is 40-fold dilution of HSB 10% acetonitrile)
HSB, 800 mL
- To 500mL ddH2O, add approx. 25-30 mL glacial acetic acid
- Slowly add 111.3 mL (80.95 g) HPLC grade triethylamine; the reaction is exothermic; the solution will become cloudy – wait until it clears before adding more)
- Add dropwise an additional 10-15 mL acetic acid to adjust the pH to 8.0.
- Add 80 mL acetonitrile (HPLC grade)
- Adjust the final volume to 800 mL with ddH2O.
LSB, 1000 mL
- To 25 mL HSB, add 100 mL HPLC grade acetonitrile
- Raise final volume to 1L with ddH2O
*Filter, and then sparge both buffers before use. Makes enough for approx. 9 runs & 2 purges.
Desalting the sample for Sep-Pack Purification
Materials:
- HPLC grade acetonitrile (a set of three runs consumes around 40 mL)
- 20 ml ddH2O, 0.22 mm filtered
- 15 mM TEAA (this can be substitutied with 0.750 mL HSB and 49.25 mL ddH2O, 0.22 mm filtered, to produce 50 mL)
- Sep-Pak cartridges, C18 (Waters #20515); 1 column per cy dye. Can be reused twice.
- 12 mL syringe (1 per dye)
Pre-load protocol:
- Wash Sep-Pak with 10 mL of acetonitrile.
- Wash with 5 mL of water.
- Wash with 10 mL of 15 mM TEAA.
Sample Preparation, Loading, and Elution:
- The column may be loaded up to 150 A260 units of sample (based on the A260 reading for both labeled and unlabeled dNTPs in the filtrate), lyophilized down to 3-5 mL (volumes of 6.5-7.5 provided reasonable yields, and volumes of u p to 10 mL have been successful, but with lower yields).
- Add 1 M TEAA to the sample, to a concentration of 15 mM.
- Load the sample onto the column. The dye should be visible near the top of the column. Wash the column with 5 mL of 15 mM TEAA.
- Elute with 3 mL of acetonitrile. Discard the initial colorless drops, collect the colored eluent into a 2 mL centrifuge tube, and discard the remaining solution. The collected volume will be approximately 1.0-2.0 mL. Some colored material will rema in on the column after 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.
HPLC Preparation
- Lyophilize the entire Sep-Pak purified material, keeping the dyes separate (see Lyophilization and Redilution section). Resuspend with a small volume (200-500 ml) of 0.2 M TEAA.
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