Annealing and extending oligos - annealing and extention of single-stranded oligo to double stranded (Mar/16/2006 )
Hi
I have a single-stranded oligonucleotide with 140mer, i dissolved it in dH2O and i want to make it double stranded with an extention primer and Klenow. Does anybody has a protocol about it?
Thanks
I don't know if it will work with an extention primer, but making single-stranded oligonucleotides double-stranded can be very easy.
Just put an ep with your oligonucleotide mix in boiling water (large volume, about 0.5L). Let it boil for 5 minutes and then let it cool down (still in the beaker with water). The trick is in the slow cooling for a good annealing.
have you both strands separately?
so you need to anneal them, withour extension.
I feel that you might design another primer annealing to other end of oligo, then simply perform PCR with the two primers, then purify product by PCR purification kit..
I have obtained dsDNA for DNA-protein binding reaction by this way.
I have obtained dsDNA for DNA-protein binding reaction by this way.
it depends from the concentration you need, but i used for yeast transormation ds oligo annealed in tis way (i did not use PPCR amplification) ant it worked.
Klenow Fragment Extension (1 nmole scale)
1. 10x Annealing Buffer: 100 mM Tris-HCl, pH 8.0 and 100 mM MgCl2. Mix 100 µL of 1 M Tris-HCl, pH 8.0 and 100 µL of 1 M MgCl2 with 800 µL Type I water. Store at -20°C.
2. 10x 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 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. Aliquot and store at -20°C. Discard if precipitated.
3. 25 mM dNTPs: Combine 250 µL each of 100 mM dATP, dCTP, dGTP and TTP. Store at -20ºC.
4. Klenow Fragment is available from Roche Molecular (Indianapolis, IN) and other suppliers.
5. Optional: [a-32P] dCTP (specific activity 3000 Ci/mmol, NEN, Boston, MA) can be used to body-label analytical reactions. Alternatively, the 5’ primer can be kinased with [g-32P] ATP (specific activity 3000 Ci/mmol, GE Healthcare ReadiVue), according to the protocol below.
6. Microcon 30 cartridges are available from Amicon (Beverly, MA).
7. Silicone oil (density 0.963, boiling point > 140°C) is available from Aldrich (Milwaukee, WI) as catalog # 14, 615-3.
1. The primer is annealed to the synthetic DNA template prior to Klenow extension. 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 mixture is vortexed and centrifuged briefly. Add 25 uL of silicone oil to prevent condensation effects. Heat the mixture at 95°C for 5 min, place the tube on ice for 5 min, then spin the contents briefly. Fast cooling results in 10% more full-length extension product than slow annealing at 2°C/min in a thermal cycler (data not shown, but this is a consistent result).
2. The annealed template and primer mixture from the previous step is used directly to set up the Klenow reaction. 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. If required for analytical purposes, add 1 µL of [a32P] dCTP (specific activity 800 Ci/mmol) to the reaction or use an end-labeled primer. Gently mix the reaction and incubate at 37°C for 30 min. Centrifuge briefly and stop the reaction by adding 12 µL of 100 mM EDTA. Do not heat inactivate, as this accelerates the 3’ > 5’ exonuclease activity. 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 ).
3. Microcon 30 (Amicon) cartridge purification: Pipet off the silicone oil. Remove the buffer and excess dNTPs by processing the entire reaction in a 30K cartridge spun at 8,160 x g at room temperature for 20 min. Rinse the filter twice with 200 µL of Type I water and spin for 8 min. Resuspend the concentrated template in 25 µL Type I water. Care must be taken not to touch the filter with a pipette tip until the sample is being withdrawn from the cartridge. Save the flow-through and wash volumes. Analyze 10 µL aliquots on analytical native acrylamide gels to be sure no product was lost.
4. Determine the concentration spectrophotometrically, assuming one A260 unit is equal to 50 µg/mL of dsDNA. Incomplete removal of dNTPs will result in values in excess of 100% conversion to dsDNA.
Comments
1. Sodium chloride is omitted from the annealing buffer in order to reduce secondary structure formation and permit the Klenow Fragment to read through regions which may self-hybridize. The buffers described in this protocol are prepared from sterilized stock solutions.
2. In general, the Klenow extension is 60-98% efficient at converting a 100 nucleotide single-stranded oligo into a completely double-stranded template. This value may vary depending upon the deprotection conditions used during oligo synthesis. There is no difference in the yields produced by normal KF and exo- KF when a 100 nuc template is used (data not shown). If larger templates are desired, the exo- version should be used.
so you need to anneal them, withour extension.
i do not have both strands actually, i have only one strand. So i try to make double stranded from a single strand DNA...