Formamide, /V-

Secondary amines do not give the expected /V-TBDMS derivatives under the conditions normally used for similar alcohol protection (Chapter 14). Instead, /V-formamides are produced (3) in good yield through a DMF-derived Vilsmeier intermediate. 

Loading solution 0.1% bromphenol blue, 0.1% xylene cyanol, 10 mAf EDTA, 95% (v/v) formamide... 

C - Cyclohexane ethylacetate water 25 75 1 D - Stationary phase 20% v/v formamide in acetone Mobile phase Chloroform etherrwater 80 20 0.5 E - Stationary phase 25% v/v formamide in acetone... 

Optimizations-. Determine which hybridization buffer composition yields the least background and strongest signal (prepare hybridization buffers consisting of 0.2% (w/v) Ficoll Type 400, 0-10x SSC in 2x increments, and 0-30% (v/v) formamide in 5% increments, see Note 6), vary incubation time and temperature (5, 10, and 20min at 23°C or 41°C is recommended), and vary liposome concentrations. 

Stationary Phase Whatman No. 1 impregnated with 40% V/V formamide in methanol. 

High-stringency buffer 50% v/v formamide, 2x SSC, lOmM DTT prepared fresh on day of use using solid DTT or a 100-mM stock of DTT stored at -20°C. 

Figure 1. Analytical anion-excbange HPLC of a commercial oligofdA) preparation on a SAX column. Sample. 10 fig oligofdA) column, Whatman Partisil 10 SAX (4.6 x 250 mm) apparatus, DuPont 850 liquid chromatograph with a DuPOnt UV spectrophotometer set at 270 nm with 0.04 AUFS (absorbance units full scale) elution, linear gtadieni from 1 mM potassium phosphate, pH 6.3, 60% (v/v) formamide to 0.3 M potassium phosphate, pH 6.3, 60% (v/v) formamide in 75 min flow-rale, 1 ml/min.

For sequences that are partially self-complementary or which have high proportions of guanine residues or more than two consecutive guanine residues eluants containing 60% (v/v) formamide are recommended (2), For the sake of convenience, we use SAX columns only with buffers containing 60% (v/v) formamide, irrespective of the composition or sequence of the oligonucleotides to be separated. A chromatogram which... 

Other anion-exchange resins have been developed which do not suffer from this drawback. We routinely now use polyethyleneimine (PEI) columns for the separation of oligonucleotides. PEI columns can be prepared by coating silica particles in situ with polyethyleneimine (4). The original protocol recommended an ammonium sulphate gradient in phosphate buffer in the presence of methanol for the separation of oligonucleotides. We run commercially available PEI wide pore columns with the same buffer system as described for the SAX columns that is solvent A, 1 mM potassium phosphate, pH 6.3, 60% (v/v) formamide and solvent B, 0.3 M potassium phosphate. pH 6.3, 60% (v/v) formamide. 

Dissolve the sample in water. Check the purity by anion-exchange HPLC, for example on a PEI column (4.6 x 250 mm) using a gradient from 0 to 70% B in 60 min at a flow-rate of 0.5 ml/min [solvent A 1 mM potassium phosphate, pH 6.3, 60% (v/v) formamide solvent B 0.3 M potassium phosphate, pH 6.3, 60% (v/v) formamide]. When the purity of the oligodeoxynucleotide is not satisfactory, carry out anion-exchange HPLC on a preparative scale using the same column under the same conditions. 

Combine two volumes of formamide loading buffer with one volume of 0.1 M NaOH. Prepare inunediately before use. The composition of formamide loading buffer is as follows 80% (v/v) formamide 10 mM EDTA 1 mg/ml xylene cyanol 1 mg/ml bromphenol blue... 

---