DNA & RNA Synthesis

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Frequently Asked Questions

How to contact us?

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DNA Sequencing & Synthesis: 
Email Core Lab  
or phone: 503-494-2472.

Microscopy: 
Email Aurelie Snyder  
or phone: 503-494-6994.

For technical assistance and admin: 
Email Tom Keller, PhD  
or phone: 503-494-2442.

Did you know?

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We can help troubleshoot your results from all Core Facility services: DNA sequencing, synthesis, and microscopy. If we make a mistake we will correct it free of charge.

Resource Links, Notes & Protocols Menu
Useful Oligo Synthesis Links FAQ & Notes  - Instrumentation  - Chemistry Notes Protocols  - Oligo Purification  - Duplex complementary strands Troubleshooting Primer Applications siRNA Synthesis

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Useful Oligo Synthesis Links

• Glen Research Additional links from Glen Research - our source for high quality reagents
• LNA monomers Probes synthesized with linked nucleic acid (LNA) monomers have higher melting temperatures for improved discrimination of exact vs mismatched sequences.
• Table of extinction coeficients Data for standard bases, base analogs and labels
• Primer3 A highly customizable primer designer from MIT
• NCBI's Gateway Website for RNA Interference Excellent primer for silencing genes with RNAi
• Essential formulae for oligonucleotides
• NCBI's RNAi Probes Resource Technical information for RNA interference experiments

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FAQ & Notes

We synthesize deoxyoligonucleotides and ribo-oligonucleotides (single-stranded DNA & RNA). Fluorescent labels, 5' terminal phosphate, biotin and many other modifications are available.

We specialize in:
• Fast turnaround: most primers are ready the next day and those marked "Rush" are ready in the morning.
• Longmers: because we use the highest quality reagents and a completely enclosed system under anhydrous argon gas, we get a better coupling efficiency than most high-throughput commercial sources. This means you get more full-length oligo. For short, primer length, oligos this may not matter.
• siRNA: 19-21 ribocucleotides with a dTdT dimer at the 3\' end is used for gene "knock down" experiments. We also synthesize all RNA, mixed DNA-RNA, and RNA with phosphorothiolate linkages.
• Fluorescent (and other) labels are very cost effective.

Instrumentation

We have two 4-column ABI 394 solid-phase DNA synthesizers. They each have 4 additional reagent ports for incorporating multiple labels, 5'-phosphate, biotin, and numerous base analogs. It is also able to synthesize RNA and mixed RNA and DNA. We are a very cost effective source for synthetic siRNA molecules.

Chemistry Notes

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Protocols

Oligo Purification

NAP-10 desalted oligos come ready to use. They're UV260 absorbance has been measured and concentration calculated from that. For most primer applications, NAP-10 desalting is sufficient.

RP-cartridge purified oligos are also quantitated and ready to use. In addition, they've had a substantial amount of failure sequence removed by the reverse-phase chromatography step. RP-cartridge purification is sometimes recommended for cell biology hybridization and mutagenesis applications.

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Duplex complementary strands

Hybridization Buffer for Annealing Complementary Oligonucleotides (e.g. duplex siRNA)

1. Prepare 100mL each of two components of Sodium Phosphate Buffer:
   Solution A - 2.76g NaH₂PO₄ monohydrate; dissolve in H₂O, volume = 100mL
   Solution B - 5.365g Na₂HPO₄*7H₂O; dissolve in H₂O, volume = 100mL
2. Combine 51mL of Solution A and 49mL of Solution B
   (this gives you 100mL of 200mM A+B at pH 6.8)
3. Prepare 200mM NaCl solution by combing 0.584g of NaCl
   in 50mL nuclease free H₂O
4. To prepare the 20mL Hybridization Buffer, add:
   10mL of 200mM NaCl
   4mL of Solution A + Solution B Mixture
   6mL of Nuclease free water

Anneal Strands

1. Combine volumes for both strands from above in one tube
2. Dry down with NO heat
3. Resuspend in 500uL Hybridization Buffer (see above recipe)
4. Heat at 90C for 3 minutes
5. Remove from heat and let cool slowly (at room temp, ~3 hours)
6. Freeze (-20C) AFTER reaching room temperature

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Troubleshooting Primer Applications

Oligo Design Problems and Possible Solutions

Performance Problem	Application	Design Remedy	Selection Featureref1
Background due to false priming in genomic or other DNA samples with much unknown sequence.	PCR, Sequencing	Select oligonucleotides with high specificiy.	Determine stability (delta G) of the 3-prime ends of oligonucleotides; select only those with low or moderate stability.
Multiple PCR product bands due to false priming within and near the intended amplificationregion.	PCR, Sequencing	Select only oligos that will not fales prime within and near the PCR product on the target DNA.	Determine the propensity of false priming using tstability (delta G) calculations; select oligos with no strong priming affinity to any other region on the target sequence.
Background due to false priming and much of the sample is known, including related available sequence files.	PCR, Sequencing	Select oligos which will not false prime on known sequence other than the target sequence	False Priming Check Against Other Sequence Files: Check for false priming sites in all the sequence files selected by the user.
High background due to false priming in repetitive sequence region in the target, e.g., ALU sequences.	PCR, Sequencing	Select oligos which will not false prime in repetitive sequence ;-)	Check for false priming sites in known repetitive sequences downloaded and selected by the user.
Low efficiency priming because of 3-prime dimerizing and hairpin loop formation of the primer itself.	PCR, Sequencing	Select oligos with low dimer and hairpin formation potential at their 3-prime ends.	Check the 3-prime ends of all oligos using stability algorithms, eliminae those with dimer or hairpin potential.

Oligo Primer Analysis Software User Manual (v.6), Section 12.3, p. 233

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siRNA Synthesis

RNA interference is the process whereby short dsRNA interferes with the cells production of the gene product. The dsRNA can be introduced by microinjection or passively (with various adjuvents) in cell culture. It can also be introduced indirectly by transfection and subsequent in vivo synthesis. Synthetic siRNA duplexes or shRNA, which get processed to siRNA, have successfully caused gene "knockdown" to >80%.This simple cartoon from MWG gives an idea the mechanism as currently understood. We synthesize the single-stranded RNA oligos ending in dTdT. We synthesize RNA at the 40 nmole and 200 nmole scale. The coupling efficiency is >98%, so you end up with >64% full-length molecule. At the 40 nmole scale, this is about a mL of ~25µM solution for each single stranded siRNA oligo. [N.B. This is a typical yield; your mileage may vary.] The reagents are expensive, but we are currently offering each 40nmole scale 21-mer siRNA (with 3'-TT) overhang for $80. For a whole lot more information on siRNA go to the NCBI siRNA website.

The most convenient way to order oligos is the on-line work request. Email the Core Facility for more information.

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