![]() ![]() ![]() The thiol group is separated to the first base by an aliphatic linker of three carbons (103 nmol): 5′-CTT-TTT-CTT-TTT-GTC-CTT-TTT-AGG-CTC-TGT-3′-(CH 2) 3-SH – Biotinylated 3-base mismatch target (184 nmol): 5′-ACA-G CG-CCT-AAA-AA C-GAC-AAA-AAG-A GA-AAG-3′-biotin.– Biotinylated target (193 nmol): 5′-ACA-GAG-CCT-AAA-AAG-GAC-AAA-AAG-AAA-AAG-3′-biotin.Both strands are biotinylated at the 3′ end for allowing hybridisation detection.Ī three-base mismatch strand with mismatches located in bases number 5, 15 and 26. Oligonucleotide solutions are prepared in TE buffer pH 8 (0.1 M Tris-HCl buffer solution, 1 mM in EDTA). Hybridisation takes place in a 2×SSC (saline sodium citrate, 30 mM sodium citrate buffer with 300 mM sodium chloride and pH 7.0) buffer containing 50% of formamide.Īlkaline-phosphatase (AP) labelled streptavidin (ST-AP) is prepared in 0.1 M Tris-HCl buffer, 1 mM MgCl 2, pH 7.2. Aliquots are prepared and maintained at –20☌. Working solutions are conserved at 4☌.ģ mM 3-indoxyl phosphate (3-IP) solutions are daily prepared in a 0.1 M Tris-HCl buffer, 10 mM MgCl 2, pH 9.8. They are kept at 4☌ and protected from light.Įthanol, magnesium chloride, sodium citrate, Trizma base, sodium chloride, EDTA, formamide, hydrochloric and sulphuric (95–97%) acids are of analytical grade. ![]()
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