1). Add 40 ul TEN buffer to the above heated reaction, place it on ice.
2). Drain a column, pipette 0.8 ml of TEN buffer onto the upper frit, and let it drain completely. Repeat this step three more times to remove the ethanol in the column completely.
3).Add the entire cDNA sample to the center øf the top frit and let it drain into the bed. Collect the effluent into tube 1.
4) Add 100 ul of Ten buffer to the column, and collect the effluent intotube 2.(Note: Let the column drain completely before the addition of new 100ul aliquøt.
5). Beggining with the next 100-aliquot of TEN buffer, collect single drop fractions into individual tubes. Continue adding 100 ul aliquots of TEN buffer until you collect a total of 18 drops into tubes 3 through 20, one drop per tube.
6) Using an automatic pipette, measure the volume in each tube; use a fresh tip for each fraction to avoid cross contamination.
7) Place the remaining tubes in a scintillation counter , and obtain Cerenkov counts for each fraction . Count the entire sample in the tritium channel without scintillation liquid.(!).
8) Measure the DNA concentration of each fraction in which the Cerenkov counts exceed background by Dip DNA kit from Invitrogen:
9).Collect cDNA from tubes 8, 9, 10, 11, and keep them individually at -70 oC to be checked for the size and cloned into a suitable vector.