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View additional product information for Taq I (TthHB8 I) - FAQs (15218019)
40 product FAQs found
If you are able to find a buffer in which all 3 enzymes have sufficient activity (usually not lower than 50%), you can set up a single digestion with all 3 enzymes. It is important that the total volume of enzymes you add to your reaction is not more than 1/10th of the total reaction volume. The reason for this is that some enzymes have star activity if the concentration of glycerol exceeds 5%. If you are not able to find a buffer in which all your enzymes have sufficient activity, you will have to perform sequential digestions of the plasmid with the individual enzymes.
Oligos should be run on a polyacrylamide gel containing 7 M urea and loaded with a 50% formamide solution to avoid compressions and secondary structures. Oligos of the same length and different compositions can electrophorese differently. dC's migrate fastest, followed by dA's, dT's, and then dG's. Oligos containing N's tend to run as a blurry band and generally have a problem with secondary structure.
Primers should be aliquoted for single use before PCR set-up. Heat just the aliquoted primers to 94 degrees for 1 min. Quick chill the primer on ice before adding to the PCR reaction. Some primers may anneal to themselves or curl up on themselves.
The drying method dries the primer in a thin layer along the sidewalls of the tube instead of the bottom, therefore a pellet is not always visible and should still be ready to use.
If the oligo was overheated, it will appear as a “ball”-shaped pellet attached to the bottom of the tube. This should not affect the quality of the oligo, and the oligo should be readily soluble in water.
If an oligo appears green in color, this is most likely due to ink falling into the tube. The oligo should still be fully functional. The color can be removed by doing an ethanol precipitation.
Most of the time the color should not affect PCR or any other experimental application since typically it is caused by the iodine used in the synthesis. There are some exceptions, however. Brown oligos can also be caused by the primer being overdried, and if this is the case, the primer may not work.
If detritylation occurs inappropriately and/or if the synthesizer has an error and delivers the wrong base, an extra inserted base can occur in your primer. Please contact techsupport@thermofisher.com for assistance.
There are two possibilities that could occur in any round of extension when creating your primer:
1.The added base is not detritylated correctly, missing one base addition but allowing possible extension in the next round.
2.The trityl group was removed, but not coupled or capped correctly before addition of the next base, allowing the chain to continue.
Better purification of the oligos is recommended to provide you with full-length oligo sequence. Adding restriction sites adds on 10 or more bases to the basic 20-25-mer, making primers longer than 30 bases with a relatively low percentage of full-length sequences after desalting. Additionally, failure sequences occur at the 5' end of the sequence as oligos are generated from 3' to 5' end. Therefore, restriction sites introduced at the 5' end of primers can be compromised, resulting in missing bases.
The oligo may not have been fully solubilized. After addition of TE buffer, make sure the oligo was vortexed for a full 30 seconds and/or pipette up and down more than 10 times. Primers may be present along the sides of the tubs, so when resuspending the oligo, the sides of the tubes should be “rinsed” too.
The scale that is ordered refers to the starting synthesis scale, or amount of starting material used to create your oligo. Based on purification and efficiency, you will receive less than the starting synthesis scale. However, we do have a minimum yield guarantee based on the starting synthesis scale which can be found here: https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-minimum-yield-guarantee.html.
This artifact occurs when either too many cycles were performed or too much DNA is added to the reaction. Try heating to 65 degrees C and putting sample on ice before loading.
Please see the following possibilities and suggestions we have:
-Primer design: try longer primers to avoid binding at alternative sites, avoid 3 consecutive G or C nucleotides at the 3' end.
-Annealing temperature: increase annealing temperature to increase specificity.
-Mg2+ concentration: try a lower concentration.
-DNA contamination: use aerosol tips and separate work area to avoid contamination, use UNG/UDG technique to prevent carryover.
Here are some reasons why your PCR experiment may be failing:
-NaCl at 50 mM will inhibit the enzyme.
-Too much KCl in the reaction. Do not exceed 50 mM.
-Incorrect annealing temperature was used.
-Incomplete denaturation (time and temperature must be long and high enough).
-Template had long runs of GC's [Woodford et al. (1995) Nucleic Acids Res 23:539 show that by eliminating all potassium from the amplification reactions, GC-rich regions in templates are sufficiently destabilized to allow PCR].
-10% DMSO partially inhibits Taq.
-Hemin (in blood samples) inhibits Taq.
-Use of super-irradiated (treated with >2500 mJ/cm2) mineral oil will either inhibit or decrease yield of PCR product [Dohner (1995) Biotechniques 18:964].
-Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that this technique can inhibit PCR [Lee (1995) BioTechniques 18:225].
-Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme). Add BSA to the PCR, increase the amount of Taq, and/or increase the volume of the PCR to dilute out them inhibitor.
Please see our suggestions below to increase yield:
-Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that this technique can inhibit PCR. [Lee (1995) BioTechniques 18:225].
-Not enough enzyme was used.
-Denaturation/extension temperature was too high and enzyme died prematurely.
-Too much DMSO (>10%).
-Incorrect annealing temperature: run a series of reactions using different annealing temperatures, starting 5 degrees below the calculated Tm.
-Too few cycles.
-Insufficient or too much Mg2+.
-Poorly designed primers: double check primer sequence against template sequence, primers should have similar melting temperatures, avoid complementary sequences at the 3' end of primers.
-Carryover inhibitors (e.g., blood, serum).
-Denaturation time was too short. Genomic and viral DNA can require denaturation times of 10 minutes.
-Not a long enough extension time was used depending on the size of product being amplified.
-Use of super-irradiated (treated with >2500 mj/cm2) mineral oil will either inhibit or decrease yield of PCR product [Dohner (1995) Biotechniques 18:964].
-Template had long runs of GC's [Woodford et al. (1995) Nucleic Acids Res 23:539 show that by eliminating all potassium from the amplification reactions, GC-rich regions in templates are sufficiently destabilized to allow PCR]. Alternatively, a combination of 1.0 M betaine with 6-8% DMSO or 5% DMSO with 1.2-1.8 M betaine can be used to amplify GC-rich templates [Baskaran (1996) Genome Res 6:633].
-Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme, melanin, etc.). Add BSA to the PCR (~160-600 µg/mL), increase the amount of Taq, and/or increase the volume of the PCR to dilute out the inhibitor. The concentration of BSA to add may be dependent on the amount and type of inhibitor present. Additionally, fatty acid-free, alcohol-precipitated BSA, or Fraction V BSA all should be effective.
Please see some reasons below for seeing smearing:
-The enzyme, primer, Mg2+, and/or dNTP concentration was too high.
-The annealing temperature was too low for the primers being used.
-Too many cycles were used.
-The annealing and extension times were too long.
-Bad or old primers.
-Too much template was used initially, try to start with 104-106 molecules
-Consider using additives or PCR Optimizer Kit (Cat. No. K122001), especially if you feel strongly that the primers should work/have worked before and are using Taq.
No, we do not guarantee 50/50 of mixed bases. If a mix of GC bases is requested, for example, the synthesizer would deliver half the normal amount of G and half the normal amount of C. Coupling efficiency is not taken into account. Therefore, it is possible that a mix, such as 30/70, will be delivered.
For 25, 50, and 200 nmol desalted and cartridge-purified DNA oligos, there is 100% A260 analysis. Random samples of 25% of the oligos produced are tested by either capillary electrophoresis or mass spectrometry. DNA oligos that are desalted and ordered at 25 and 50 nmol scales also have 100% real-time digital trityl monitoring during analysis. Desalted DNA oligos ordered at 1 and 10 µmols, DNA oligos at any scale that are purified by HPLC and PAGE, the majority of the DNA oligos with 3' and/or 5' modifications, and RNA oligos have 100% A260 analysis and capillary electrophoresis or mass spectrometry.
The plate orders must contain an average of 24 or more oligos per plate for 96-well plates or 192 or more oligos per plate for 384-well plates across the entire order.
Tm values are not absolute - they are an approximation of the melting temperature range which exists. A thermal profile for a given oligo shows a 10-15 degree range of melting depending on the amount of salt but also on the base composition and concentration of primer in the reaction which are not precisely defined. One should not rely solely on the given Tm value as the only one that will work. Tm is the temperature at which 50% of the primer and its complementary sequence are present in a duplex DNA molecule. The Tm is necessary to establish an annealing temperature for PCR. Reasonable annealing temperatures range from 55 degrees C to 70 degrees C. Annealing temperatures are generally about 5 degrees C below the Tm of the primers. Since most formulas provide an estimated Tm value, the annealing temperature is only a starting point. Specificity for PCR can be increased by analyzing several reactions with increasingly higher annealing temperatures.
As oligos increase in length, the column purification is less effective in separating the failure oligos from the correct products. PAGE purification would be the method of choice in this case.
Value Oligos are the most cost-effective and fastest way to order oligos. They are available for 5-40-mers, at a 25 or 50 nanomole scale, with a range of purification options to suit your needs, and are eligible for next-day delivery. The cost is calculated per oligo as opposed to per base. Value Oligos are not available with modifications. Value Oligos undergo the same QC standards as our standard oligos with the same manufacturing process.
A common equation used to calculate primer Tm is as follows: Tm (in degrees C) = 2 (A+ T) + 4 (G + C)
Please take a look at this list (https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-modification-options.html) of standard modification options that we offer. If you do not see the modification option you would like, please email our Technical Support team at techsupport@thermofisher.com to see if we can accommodate your request.
The percentage of full-length oligonucleotide depends on the coupling efficiency of the chemical synthesis. The average efficiency is close to 99%. To calculate the percentage of full-length oligonucleotide, use the formula: 0.99n-1. Therefore, 79% of the oligonucleotide molecules in the tube are 25-bases long; the rest are <25 bases. If you are concerned about starting with a preparation of oligonucleotide that is full-length you may want to consider cartridge, PAGE, or HPLC purification.
Coupling efficiency is important as the effects are cumulative during DNA synthesis. The numbers below shows the effect of a 1% difference in coupling efficiency and how this influences the amount of full-length product available following synthesis of different length oligos. Even with a relatively short oligo of 20 bases, a 1% difference in coupling efficiency can mean 15% more of the DNA present following synthesis is full-length product.
Number of bases added, 99% coupling full-length, Failures, 98% coupling full-length, Failures:
- 1, 99, 1, 98, 2
- 2, 98.01, 1.99, 96.04, 2.96
- 3,97.03, 2.97, 94.12, 5.88
- 10, 90.44, 9.56, 81.71, 18.29
- 20, 81.79, 18.21, 66.76, 33.24
- 30, 73.79, 26.03, 54.55, 63.58
- 50, 60.5, 39.5, 36.42, 63.58
- 95, 38.49, 61.51, 14.67, 85.33
The scale of synthesis is the starting point for synthesis, not the guaranteed final amount. We guarantee the total yield of oligonucleotide as a minimum number of OD units. Use this link (https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-minimum-yield-guarantee.html) for the minimum yield guarantees we offer for our oligos.
Yes. OligoPerfect Designer can be used to design primers for sequencing, cloning, or detection.
These guidelines may be useful as you design your PCR primers:
- In general, a length of 18-30 nucleotides for primers is good.
- Try to make the melting temperature (Tm) of the primers between 65 degrees C and 75 degrees C, and within 5 degrees C of each other.
- If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.
- Aim for the GC content to be between 40 and 60%, with the 3' of a primer ending in C or G to promote binding.
- Typically, 3 to 4 nucleotides are added 5' of the restriction enzyme site in the primer to allow for efficient cutting.
- Try to avoid regions of secondary structure, and have a balanced distribution of GC-rich and AT-rich domains.
- Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT).
- Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.
- If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification.
- If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.
- If you are using the primers for a PCR reaction to be used in TOPO cloning, the primers should not have a phosphate modification.
Read more about primer design tips and tools at https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/primer-design-tools.html.
You may choose to do a two-temperature protocol when the annealing temperature is relatively high. In this case, you would combine the annealing and the elongation steps, i.e., both can occur together at a temperature >62 degrees C. The advantage of a two-temperature protocol is that it is considerably quicker in comparison to the conventional three-temperature protocol.
You can try adding 5-10% DMSO, up to 10% glycerol, or 1-2% formamide or a combination of these to facilitate difficult templates. Note: the use of cosolvents will lower the optimal annealing temperatures of your primers.
A GC-rich template often has a higher melting temperature and may not denature completely under the normal reaction conditions.
Hot start is a way to prevent DNA amplification from occurring before you want it to. One way to do this is to set up the PCR reaction on ice, which prevents the DNA polymerase from being active. An easier method is a use a ‘hot-start' enzyme, in which the DNA polymerase is provided in an inactive state until it undergoes a high-heat step.
The main steps are: denaturation, annealing, and extension. The template is typically heated to a high temperature (around 94-95 degrees C) allowing for the double-stranded DNA to denature into single strands. Next, the temperature is lowered to 50-65 degrees C, allowing primers to anneal to their complementary base-pair regions. The temperature is then increased to 72 degrees C, allowing for the polymerase to bind and synthesize a new strand of DNA.
Please see the comparison below on the following criteria:
Enzyme, Relative Fidelity, Amplicon Length, and 3' Overhang (+/-)
Taq, 1, <5 kb, +
Platinum II Taq Hot-Start, 1, <5 kb, +
Platinum Taq, 1, <5 kb, +
AccuPrime Taq, 2, <5 kb, +
Platinum Taq HiFi, 6, <20 kb, +/-
AccuPrime Taq HiFi, 9 <20 kb, +/-
Platinum Pfx, 26, <12 kb, -
AccuPrime Pfx, 26, <12 kb, -
Pfx50, 50, <4 kb, -
AmpliTaq, 1, <5 kb, +
AmpliTaq Gold, 1, <5 kb, +
AmpliTaq Gold 360, 1, <5 kb, +
Taq error rate: 1 x 10-4 to 2 x 10-5 base/duplication
Certain restriction enzymes are unable to recognize and cleave at their target sites if specific adenine or cytosine residues in the sequence are methylated, and Dam and Dcm are two E. coli methylases which introduce methyl groups that affect the cutting sites of many common enzymes. The methylase encoded by the dam gene (Dam methylase) transfers a methyl group from S-adenosylmethionine to the N6 position of the adenine residues in the sequence GATC. The Dcm methylase (encoded by the dcm gene; referred to as the Mec methylase in earlier references) methylates the internal cytosine residues in the sequences CCAGG and CCTGG at the C5 position.
To take advantage of Dam- and Dcm-sensitive restriction enzymes and get proper cleavage, plasmid DNA must be propagated in and isolated from an E. coli strain that is deficient in the endogenous Dam methylase and Dcm methylase enzymes just prior to the restriction reaction. We have one competent cell product available that is made with a dam- and dcm- strain: One Shot INV110 Chemically Competent E. coli (Cat. No. C7171-03).
Here are some recommendations:
1. You can verify the restriction endonuclease has activity by digesting the unit substrate (i.e., lambda or Ad-2 DNA) using the reaction conditions identified for unit definition: one unit of the restriction endonuclease should digest one µg of the unit substrate in one hour in the appropriate reaction buffer at the appropriate temperature.
2. You can test for the presence of inhibitors of restriction digestion in the sample DNA. Perform a restriction digest in which some of the sample DNA is included along with some of the unit substrate (i.e., lambda or Ad-2 DNA). If digestion of the unit substrate occurs alone but is not observed when the sample DNA is added, then a diffusible inhibitor of restriction digestion is present in the sample DNA. If digestion of the unit substrate occurs in the presence of the sample DNA, but the sample DNA is not digested, then the failure of the restriction endonuclease may be due to sensitivity of the restriction endonuclease to methylation in the sample DNA.
3. Finally, to identify any tube-specific issues like shipment or storage stability problems, you can test function by performing a side-by-side reaction with a different lot of the same Invitrogen restriction endonuclease and comparing results.
Three major sources of problems are described below:
1. The DNA isn't clean enough to cut. Contaminating by-products from cells and purification chemicals can inhibit restriction enzymes. We recommend using high purity DNA isolation method like those found in our Purelink plasmid isolation products, or an equivalent protocol such as two phenol-chloroform extractions with isopropanol precipitations in between. See below for a suggested protocol.
2. The DNA may be heavily methylated. This can be checked by cutting with 4-base enzymes like HhaI, MboI, HpaII, etc. They should cut something - if they do not, that site is methylated. You can also check by cloning a piece of DNA and seeing if it now has the sites that the genomic DNA did not have.
3. The DNA may be very high in GC or very high in AT, and either extreme results in very little cutting by enzymes like EcoRI and HindIII. It is hard to tell the difference between very little cutting and no cutting with genomic DNA. This also can be tested by cutting with 4-base enzymes like HpaII (CCGG), HhaI (GCGC), MseI (TTAA) and DraI (TTTAAA). It might be better to do partial cuts with a 4-base cutter that will cut many times than to try to cut with a 6-base enzyme with more rare sites. Match enzymes and cloning sites, e.g. HpaII/ClaI, MseI/EcoRI, Sau3aI/BamHI.
Suggested protocol to purify plasmid from 200-400 mg cells:
1) Resuspend cells in 10 ml TE-lysozyme
2) Incubate 5 min at RT
3) Add 50 ml 10 mg/ml Proteinase K
4) Add 10 ml 1% SDS in TE
5) Vortex for 30 sec
6) Place at 45°C for 30 min or until clear
7) Vortex for 10 sec Can store overnight
8) Add 2 ml 3 N NaOAc
9) Add 20 ml phenol-CHCl3
10) Vortex 10 sec
11) Spin 20 min @ 3000-4000 RPM
12) Carefully remove up to 15 ml of supernatant (do not take milky interface) and place in 50 ml conical tube
13) Add 9 ml isopropanol
14) Invert several times to mix. Do not vortex. Precipitate should form wad.
15) Centrifuge 5 min @ 3000-4000 RPM or pick out with pipet
16) Wash pellet in 5 ml 70% EtOH
17) Resuspend pellet in 10 ml TE + 10 mg/ml RNAseA - place in 2 polypropylene 15ml conical tubes for 5 min 60 C
18) Add 0.5 ml 3 N NaOAc to each tube
19) Vortex 10 sec
20) Add 5 ml phenol/chloroform to each tube
21) Vortex 10 sec
22) Centrifuge 20 min @ 3000-4000 RPM
23) Remove 4 ml supernatant from each tube and place in 15 ml conical tube
24) Add 4.8 ml EtOH
25) Invert several times to mix. Precipitate should be a smaller single wad
26) Centrifuge or pick out precipitate
27) Wash in 1 ml 70% EtOH
28) Resuspend in 1 ml TE. Thick, very viscous, gel-like: 1 mg/ml. Dilute 2X. Very viscous, but clear solution: about 500 mg/ml. Slightly viscous, detectable when hand vortexing: about 100 mg/ml.
29) Place in tube marked with date. Store at -20°C for temporary storage, and -80°C for long term.
Please use our DoubleDigest Calculator linked below: https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/double-digest-calculator-thermo-scientific.html