[分享]oligo退火的条件

Ambion公司的protocol:将合成的DNA(长度约55nt)溶解成1ug/ul,各取2ul,加46ul退火缓冲液(100mM乙酸钾，30mMHEPES-KOH pH7.4,2mM乙酸镁)，90度30min,37度1h,然后可以用来联接，或贮存于-20度。我们实验室有人按此方法成功了,我正在做。其他的非RNAi专用的退火条件有：TaKaRa:1.用STE缓冲液(10mM Tris pH8.0,50mM NaCl, 1mM EDTA)溶解DNA，浓度1-5OD/100ul2.等摩尔混合3.94度5min,徐冷到室温4.供实验用Sigma:Annealing Buffer: 10mM Tris, pH 7.5 - 8.0, 50mM NaCl, 1mM EDTA1xTE Buffer: 10mM Tris, pH 7.5 - 8.0,1mM EDTA1. Resuspending the Oligonucleotides: Resuspend both complementary oligonucleotides at the same molar concentration, using Annealing Buffer (see note below). For convenience, keep Annealing Buffer volume below 500ul for each oligo. Annealing should perform well over a wide range of oligo concentrations. For larger scale oligo syntheses, it may be necessary to use larger volumes that can be aliquoted after resuspension.2. Annealing the Oligonucleotides: Mix equal volumes of both complementary oligos (at equimolar concentration) in a 1.5ml microfuge tube. Place tube in a standard heatblock at 90 - 95ºC. Remove the heatblock from the apparatus and allow to cool to room temperature (or at least below 30ºC) on the workbench. Slow cooling to room temperature should take 45-60 minutes. Store on ice or at 4ºC until ready to use. An alternative procedure for annealing involves the use of a thermal cycler. Dispense 100ul aliquots of the mixed oligos into PCR tubes (500ul size). Do not overlay the samples with oil. Place the tubes in a thermal cycler and set up a program to perform the following profile: (i) heat to 95ºC and remain at 95ºC for 2 minutes, (ii) ramp cool to 25ºC over a period of 45 minutes, (iii) proceed to a storage temperature of 4ºC. Briefly spin the tubes in a microfuge to draw all moisture from the lid. Pool samples into a larger tube, store on ice or at 4ºC until ready to use.3.Long Term Storage. It may be necessary to aliquot and lyophilize the annealed sample. After drying, the sample may be stored at -20ºC in a desiccated container. Resuspend the annealed oligos at the desired concentration with sterile distilled water. The annealed pair of oligonucleotides is ready for use.NOTE: Oligos may also be resuspended in either 1x Ligase Buffer or 1x Kinase Buffer instead of the above Annealing Buffer (prior to annealing).SBI：• 2X DNA Annealing Buffer(20mM Tris, pH 7.8; 100mM NaCl; 0.2mM EDTA)Anneal siRNA Oligonucleotidesa. Dissolve the siRNA oligonucleotides in an appropriate amountfinal concentration of 1 µg/µl.b. Prepare the ds siRNA oligonucleotide as follows:2.5 µl Sense strand siRNA oligonucleotide2.5 µl Anti-sense strand siRNA oligonucleotide25.0 µl 2X Annealing Buffer20.0 µl Deionized water50.0 µl Total volumec. Heat the mixture to 95°C for 5 min in a thermocycler or heatingd. Turn off the thermocycler or heating block and let it cool toe. The annealed oligonucleotide (100 ng/µl) is ready for phosphorylationStore the annealed oligonucleotides at -20°C until use.上面的英文就不一一翻译了。TaKaRa和SBI的方法我用过，但没有联接成功：原因正在寻找，可能是我的载体的问题，感受态细胞的问题，也可能是退火的问题，还有可能是DNA合成的问题，等等。God bless me!编辑：wudihero007