How much of the first-strand reaction should I add to the PCR?

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Answer

The volume of the first-strand reaction to add to your PCR reaction will depend on the starting amount of RNA used for first-strand synthesis, and the abundance of the target gene. We recommend starting with 10% of the first-strand reaction. More than 10% may inhibit the PCR.

Answer Id: E2927

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c46f627c58698723138074c39f423b68_FAQ

What is the highest temperature that SuperScript® III , SuperScript II®, MMLV, or ThermoScript™ can be used?

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Answer

The optimal temperature for SuperScript® III RT is 50 degrees C, and can be used up to 55 degrees C. For some qRT-PCR reactions where gene-specific primers are used, you can do the RT reaction at 60 degrees C. The optimal temperature for SuperScript® II RT is 42 degrees C, and can be used up to 50 degrees C. Optimal temperature for MMLV is 42 degrees C. ThermoScript™ RT shows optimal activity at 60 degrees C, and can be used at temperatures as high as 70 degrees C (for amplicons expected to be 1 kb or less). For PCR products expected to be greater than 1 kb, a maximum first strand synthesis temperature of 60-65 degrees C is suggested. Be sure your first-strand primer anneals at the high temperature, especially when gene-specific primers are used for high-temperature stable reverse transcriptases. We recommend oligo (dT)20 for cDNA synthesis when using an oligo (dT) primer for first-strand synthesis with these enzymes.

Answer Id: E2928

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d7c3ba7384975fef7db5edd4b74715ea_FAQ

What are the best conditions for double digests with restriction endonucleases?

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Answer

First, please note that we changed our restriction enzymes and buffer formulations in 2010. The REact® buffers 1-10 were discontinued in favor of a smaller group of universal buffers: H, K, L, M, T. The new buffers are not compatible with older restriction enzymes, and it is not recommended to do a double digest with an old enzyme (with REact® buffer) and a new enzyme.

When performing any double digest, there may be buffer incompatibilities and enzyme steric hindrance problems. These can be avoided by performing sequential digests, separated by buffer exchange or chloroform extraction and ethanol precipitation. However, if these issues are understood and a double digest will be performed, you can evaluate enzyme combinations using our buffer compatibility chart. You can find the chart by searching “Recommended universal buffers for double digestion” on the Life Technologies website.

Answer Id: E2929

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5c624e4c6fc297da3f273936e644fb20_FAQ

What type of modified primer bases do you sell?

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Answer

Primers can contain deoxyuracil (at any position except the 3’ end), deoxyinosine (at any position except the 3’ end), phosphorothioates, and mixed bases (2, 3, or 4 equimolar degenerate bases). Also, various 5’ or 3’ modifications are available through our Custom Primer service. For more information, please see "Oligos, TaqMan® Probes & Primers" under Products & Services on our website.

Answer Id: E2930

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4541494203cf99c37d67ab3a6a24fbab_FAQ

How are the primers synthesized?

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Answer

Oligos are made using an DNA synthesizer, which is basically a computer-controlled reagent delivery system. The first base is attached to a solid support, usually a glass or polystyrene bead, which is designed to anchor the growing DNA chain in the reaction column. DNA synthesis consists of a series of chemical reactions.

(1) Deblocking
The first base, attached to the solid support via a chemical linker arm, is deprotected by removing the trityl protecting group. This produces a free 5’ OH group to react with the next base.

(2) Coupling
The next base is added, which couples to the first base.

(3) Capping
Any of the first bases, which failed to react are capped. These failed bases will play no further part in the synthesis cycle.

(4) Oxidation
The bond between the first base and successfully coupled second base is oxidized to stabilize the growing chain.

(5) Deblocking
The 5’ trityl group is removed from the base, which has been added.

Each cycle of reactions results in the addition of a single DNA base. A chain of DNA bases can be built by repeating the synthesis cycles until the desired length is achieved.

Answer Id: E2931

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d7917e7277de0ed668a5e3a3c862b68e_FAQ

What scales of primer synthesis are offered and which one should I choose?

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Answer

For Invitrogen™ Custom DNA Oligos, we offer 25 nmol, 50 nmol, 200 nmol, 1 μmol, and 10 μmol starting scales of synthesis. A 25 nmol scale will give a minimum yield of 2 O.D. with desalted purity. A 50 nmole starting scale of synthesis of a non-modified primer yields 2 to 4 O.D. units (depending on the size and purity of the primer). A similar 200 nmol scale of primer yields between 8 to 20 O.D. units and a 1 μmol scale would yield 20 to 50 O.D. units. A similar primer ordered at the 10 µmol scale would yield 200 to 500 O.D. units. The yield from a 50 nmol starting scale generally is sufficient for 1,000 to 5,000 PCR reaction (assuming the PCR reaction is a 100 μl and the primer concentration was 100 to 500 nM).

Answer Id: E2932

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8e7127881889c82e45004a0bc2d9c595_FAQ

What primer purification options are available, and how should I choose between them?

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Answer

Invitrogen™ Custom DNA Primers are offered with several options for primer purification. Which one you should choose depends on your application. Here are some details on the options that are typically available:

Desalted--this is our standard purification procedure. The primer will be gas-phase deprotected and normal-phase desalted. Organic products are removed with 10% acetonitrile/90% water wash, and the products are then eluted under aqueous conditions. The smallest oligo we synthesize at this purification is 5 bases. Desalting removes salt, but not failure sequences. This method is sufficient for standard PCR, RT-PCR, cDNA synthesis, and sequencing reactions.

Cartridge Purification--this is based on reversed-phase chromatography to provide full-length sequences. The majority of the n-1, n-2, n-3, etc. species will be removed for oligos less than 35 bases. The smallest oligo we synthesize at this purification is 7 bases. Cartridge Purification is recommended for PCR with primers that have critical 5' sequences (RE sites, promoters), site-directed mutagenesis, first-strand cDNA synthesis for generation of libraries, gel shift assays, and production of cloning adapters.

HPLC (high-pressure liquid chromatography)--HPLC is a reversed-phase chromatography method similar to that used in cartridge purification. This process removes failure sequences and unincorporated labels. This is offered for oligos up to 50 bases long. HPLC is recommended for antisense work.

PAGE Purification--a denaturing polyacrylamide gel is used to purify the full-length primers from ALL types of n-1, n-2, etc. species of oligos. Oligos are purified by electroelution from the acrylamide gel and then run on a desalting column. These primers are not phenol extracted. PAGE purification can be used for any length of oligonucleotides. Typical yields for PAGE purified primers are not as high as when purified by other methods, but the product is highly pure. This is recommended for GeneTrapper screening. For more information, refer to the Oligos pages under Products & Services on our website.

Answer Id: E2933

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b37733f7ef24fd86b05d5f4a0dcda8f9_FAQ

How are custom primers packaged?

Product FAQ

Answer

Primers are lyophilized and shipped at room temperature in a 2 mL screw cap, clear, polypropylene tubes. Large orders (over 48 primers) may be arrayed in 1-mL 96-well polypropylene plates. Fluorescent-labeled primers are supplied in amber screw cap polypropylene tubes. The alkaline phosphatase- and horseradish peroxidase-labeled primers are supplied in solution in a storage buffer unique to the enzyme label.

Answer Id: E2935

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09f58d264dc48bb499744472e1f5580c_FAQ

What information is provided with a custom primer?

Product FAQ

Answer

A Certificate of Analysis is provided for each custom primer, which includes: primer name; sequence and length; average coupling efficiency; melting temperature; GC content; amount in ODs, μg, and nmole; and molecular weight and extinction coefficient calculated from the sequence.

Answer Id: E2936

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f8b7de242d39e5c227b495d31f5e520b_FAQ

How should I reconstitute the oligonucleotide? How long can the oligonucleotide be stored?

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Answer

Reconstitute the oligonucleotide by resuspending the oligonucleotide in TE [10 mM Tris-HCl (pH 8.0), 1 mM EDTA] at a concentration more than or equal to 10 μM. The lyophilized oligonucleotide is stable at -20 degrees C for 1 year or more. The oligonucleotide dissolved in TE is stable for at least 6 months at -20 degrees C or 4 degrees C. Oligonucleotides dissolved in water are stable for at least 6 months at -20 degrees C. Do not store oligonucleotides in water at 4 degrees C. TE is recommended (instead of deionized water) since the pH of the water is often slightly acidic and can cause hydrolysis of the oligonucleotide.

Here is the calculation for reconstitution: Find nmoles of primer from Certificate of Analysis or tube label. 1 nmole = 1,000 pmoles 1 μM = 1 pmole/μl Therefore, to make a 20 μM solution from 4 nmoles of primer, 4,000 pmoles/20 pmoles per μl = 200 μl TE buffer.

Answer Id: E2937

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94cf6898661f9efad7cff9c17773b255_FAQ

What conditions are optimal for fill-in reactions using Klenow (large fragment of DNA Polymerase)?

Product FAQ

Answer

Klenow can be used to "fill in" 5' overhangs of double-stranded DNA fragments using common restriction endonuclease buffers. We recommend REact 2 buffer (1X concentration is 50 mM Tris-HCl pH 8, 50 mM NaCl, 10 mM MgCl2) , but other buffers will also work.

Fill-in Reaction Conditions:
1. Dilute Large Fragment of DNA Polymerase I to 0.5 U/μl with Klenow Dilution Buffer.
2. To a 1.5-ml microcentrifuge tube on ice, add: 10X REact 2 Buffer - 3 μl, 0.5 mM dATP - 1 μl, 0.5 mM dCTP - 1 μl, 0.5 mM dGTP - 1 μl, 0.5 mM dTTP - 1 μl, DNA 0.5-1 μg, Large fragment of DNA Polymerase I - 1 μl, Autoclaved distilled water to 30 μl.
3. Mix gently and centrifuge briefly to bring the contents to the bottom of the tube.
4. Incubate at room temperature for 10-15 minutes or 20 minutes on ice.
5. Terminate fill-in reaction by phenol extraction.
To label the DNA fragment, use 1-2 μl of [α-32P]dNTP (400 Ci/mmol, 10 mCi/ml) (24-48 pmoles) instead of the corresponding cold dNTP.
Life Technologies™ also supplies an Exo minus Klenow, which is provided with its own buffers.

Answer Id: E2939

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e20d712ee4ca0a25abe38ad04e4a7e36_FAQ

Why is ATP present in the reaction buffer for T4 DNA Ligase?

Product FAQ

Answer

ATP is necessary for enzymatic function. It is involved in phosphorylating the ligase prior to the ligation reaction. Ligation efficiency is markedly reduced by removing ATP from the reaction. It is important, therefore, to handle the buffer appropriately in order to minimize degradation of ATP.

Answer Id: E2940

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151fa50c3e2bee6933194675d6f6e71b_FAQ

What are the recommended conditions for ligating single-stranded RNA fragments?

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Answer

The following conditions will enable you to join two oligonucleotides with T4 RNA ligase:
50 mM HEPES (pH 8.3), 3 mM DTT, 10 μg/ml BSA, 10 mM MgCl2, 0.5 mM acceptor RNA, 0.6 mM donor RNA, 2 mM ATP, 180 μg/ml RNA ligase.

10-20% DMSO can be added to stimulate activity. Incubate 10 h at 16°C.

Answer Id: E2941

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e0c2c1118939f2d32fd8b671752eaff4_FAQ

Are your DNase I products RNase-free?

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Answer

Most of our DNase I products are guaranteed free of RNase activity. However, please note that product 18047-019 is not tested for RNAse and is recommended primarily for protein applications. The other products are suitable for removing DNA from both RNA and protein preparations, for nick translating DNA, and for generating random fragments of DNA. For more demanding RT-PCR applications, we recommend using DNAse I, Amplification Grade.

Answer Id: E2942

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808eac3cdf97d0f48a0bf43c48ce2dbd_FAQ

What is the specific activity of your DNase I products, SKU # 18047019 and 18068015?

Product FAQ

Answer

Specific activities vary by lot. Our DNase I must have a specific activity greater than 10,000 units/mg to pass our quality standards.

Answer Id: E2943

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b5f40a66ad37749bdbee9701a9021d6e_FAQ