Klenow fragment extension11/12/2023 ![]() ![]() Microcon 30 (Amicon) cartridge purification: Pipet off the silicone oil. This protocol should generate 1000-1500 nmoles of completely double-stranded DNA template (the remaining 200-700 pmoles is a ladder of partially extended products ).ģ. Do not heat inactivate, as this accelerates the 3’ > 5’ exonuclease activity. Centrifuge briefly and stop the reaction by adding 12 µL of 100 mM EDTA. Gently mix the reaction and incubate at 37Â☌ for 30 min. ![]() If required for analytical purposes, add 1 µL of dCTP (specific activity 800 Ci/mmol) to the reaction or use an end-labeled primer. Mix the components in the order listed in a microfuge tube on ice: 90 µL of annealed template/primer, 30 µL of 10x Klenow Fragment buffer, 7 µL of 1 M MgCl2, 34 µL of 25 mM dNTPs (final concentration of 0.85 mM dNTP for a 100 nuc oligo), 45 units of Klenow Fragment and Type I water to a final volume of 300 µL. The annealed template and primer mixture from the previous step is used directly to set up the Klenow reaction. Fast cooling results in 10% more full-length extension product than slow annealing at 2Â☌/min in a thermal cycler (data not shown, but this is a consistent result).Ģ. Heat the mixture at 95Â☌ for 5 min, place the tube on ice for 5 min, then spin the contents briefly. Add 25 uL of silicone oil to prevent condensation effects. The mixture is vortexed and centrifuged briefly. In a siliconized microfuge tube, mix 9 µL of 10x annealing buffer with 2 nmol of gel-purified 5’ primer and 1.7 nmol of gel-purified template (a 1.2:1 ratio of primer:template) and adjust the volume to 90 µL with water. The primer is annealed to the synthetic DNA template prior to Klenow extension. Silicone oil (density 0.963, boiling point > 140Â☌) is available from Aldrich (Milwaukee, WI) as catalog # 14, 615-3.ġ. Microcon 30 cartridges are available from Amicon (Beverly, MA).ħ. Alternatively, the 5’ primer can be kinased with ATP (specific activity 3000 Ci/mmol, GE Healthcare ReadiVue), according to the protocol below.Ħ. Optional: dCTP (specific activity 3000 Ci/mmol, NEN, Boston, MA) can be used to body-label analytical reactions. ![]() Klenow Fragment is available from Roche Molecular (Indianapolis, IN) and other suppliers.ĥ. Discard if precipitated.ģ.Ē5 mM dNTPs: Combine 250 µL each of 100 mM dATP, dCTP, dGTP and TTP. Mix 5 mL of 1 M Tris-HCl, pH 7.4, 1 mL of 1 M MgCl2, 10 µL of 1 M DTT and 500 µL of 10 mg/mL BSA with 3.5 mL of Type I water. Store at -20Â☌.Ģ.đ0x Klenow buffer: 500 mM Tris-HCl, pH 7.4, 100 mM MgCl2, 1 mM dithiothreitol (DTT) and 500 µg/mL Bovine Serum Albumin (BSA). Mix 100 µL of 1 M Tris-HCl, pH 8.0 and 100 µL of 1 M MgCl2 with 800 µL Type I water. Klenow Fragment Extension (1 nmole scale)ġ.đ0x Annealing Buffer: 100 mM Tris-HCl, pH 8.0 and 100 mM MgCl2. ![]()
0 Comments
Leave a Reply.AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |