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Polymerase
1. MgCl2 concentration
3B polymerase uses 2 mM MgCl2. It is most important to optimise this concentration for each experiment, in order to obtain the maximum yield.
2. Why two concentrations?
Some experiments require the use of small amounts of polymerase. In order to avoid pipetting errors, 1 U/µl is used. For experiments where volumes of polymerase are not so small, or final reaction volume is critical (e.g. microwell plates), then we recommend to use 5 U/µl.
3. How much enzyme has to be used?
It depends on each experiment. For standard amplifications, 1 unit / 50 µl final reaction volume is enough. For long PCRs (products longer than 2 Kbp), PRINS, in situ PCR, or PCRs of more than 30 cycles, it may be necessary to add more enzyme (from 2 units up to 4 units / 50 µl final reaction volume).
4. Why two buffers?
Standard buffer has MgCl2 included in it, at a concentration of 2 mM. It is the usual buffer of choice for routine applications. However, if the experiment has to be optimised, we recommend to use Mg-free buffer,and optimise the MgCl2 accordingly.
5. Can the amplification product be used in T/A cloning?
Yes, it can. The polymerase leaves A-ends in the amplification products, thus they can be directly used in T/A cloning. Only make sure to use the correct T/A cloning kit (one leaving T-ends, not blunt ends).
6. Thermostability
The polymerase activity is not affected after more than 40 cycles of amplification. Its half life at 94 ºC is 40 min.
7. Long PCRs
The polymerase has been tested in PCRs up to 5 Kbp. Some reports by customers indicate successful results in PCRs of up to 10 Kbp. However, it must be reminded that fidelity is crucial in long PCRs, thus the customer has always to be reminded about the possibility of combining our polymerase with proof-reading enzymes (1 unit of BT pol per 0.1 units of proof-reading enzyme), if fidelity is important.
For long PCRs, we recommend to increase the amount of polymerase (from 2 units to up to 4 units), the amount of dNTPs (250-300 µM final concentration), and the elongation times (1 min/Kbp to be elongated). Some increase in MgCl2 concentration may also be necessary.
8. Amplifications involving bacterial DNA, RAPDs
3B DNA Polymerase is NOT recommended for non-stringent applications, such as RAPDs, or amplifications involving sequences homologous to E. coli. Recommend use of ULTRATOOLS for these applications.
9. Storage
The 3B DNA Polymerase can endure room temperature for up to three weeks. Dry ice is used more as a way of maintaining an image than as really necessary.
Polymerase can never be frozen. This does not inactivate the enzyme, but dramatically decreases its activity. Customers storing the polymerase at - 70ºC, or in frost-free freezers, must discard the polymerase.
10. High GC content in template DNA
3B  polymerase  has a high yield when using difficult templates (e.g. high GC content). We only recommend to increase denaturation times up to 1 minute, and perform an initial denaturation of up to 10 minutes. BT pol can endure these long incubations at high temperature without losing its activity.
A higher Mg concentration is also recommended.
11. Low yield
The first question that must always be made: has Mg concentration been optimised? If the concentration has been optimised, further investigation must be made on the experiment in order to know what has happened. Do not hesitate to contact us for further technical assistance, e-mails, phone calls, faxes .

 

Retrotools
1. MgCl2 concentration
3B RETROTOOLS uses 2 mM MgCl2. For experiments needing to optimise this concentration, a Mg-free format is available.
2. Why two buffers?
The enzyme included in RETROTOOLS kit has reverse transcriptase activity in the presence of Mn2+ ions, while in the presence of Mg2+ ions, the activity switches to polymerase. RT buffer must be supplied with MnCl2 in order to optimise its activity, while DNA buffer, apart from having MgCl2 (in Mg-included formats), has a chelating agent that blocks Mn2+ ions, allowing the enzyme to polymerise.
3. Can the amplification product be used in T/A cloning?
Yes, it can. The polymerase activity leaves A-ends in the amplification products, thus they can be directly used in T/A cloning. Only make sure to use the correct T/A cloning kit (one leaving T-ends, not blunt ends).
4. Thermostability
The polymerase activity is not affected after more than 40 cycles of amplification. Its half life at 94 ºC is 40 min. Reverse transcription can be performed in a wide range of temperatures, from 45 ºC to 70 ºC (or up to  75 ºC, according to customers' reports).
5. Long RT-PCRs
3B RETROTOOLS can reverse transcribe and polymerise RNAs of up to 1 Kbp. For longer products, we do not recommend the use of this kit.
6. Storage
3B RETROTOOLS can endure room temperature for up to two weeks.
The enzyme can never be frozen. This does not inactivate the enzyme, but dramatically decreases its activity. Customers storing the polymerase at - 70ºC, or in frost-free freezers, must discard the kit.
7. Low yield
On some occasions, and for customers using 3B RETROTOOLS from time to time, some precipitate can appear in the buffers. It is of the utmost importance to shake well all vials before proceeding with the reaction. If this is not enough to eliminate the precipitate, then an incubation of 10 min at 65 ºC usually eliminates it. For rare messengers, a cyclic process can increase the obtained yields (see leaflet).
Ultratools

Questions are very much similar to those in 3B DNA Polymerase. Other specific questions for ULTRATOOLS are:
1. Amplifications involving bacterial DNA

We recommend using ULTRATOOLS. This does not mean that 3B DNA polymerase cannot perform them. However, we cannot guarantee that 3B DNA Polymerase is completely free of genomic DNA from E. coli. Though in 90 % of the cases this genomic DNA does not interfere (and negative controls render no bands), there have been around 7 customers worldwide who have had problems in this sense. So we recommend, as a cautionary measure, to use ULTRATOOLS, where absence of genomic DNA from E. coli is guaranteed.
2. Amplifications involving non-bacterial DNA
3B ULTRATOOLS is applied in these applications where annealing temperature is low (e.g. RAPDs) or where sequences homologous to those found in E. coli are used. Sequences homologous to E. coli may be known in advance or not (known in advance: primers specifically designed to anneal with E. coli sequences. Not known in advance: primers designed to anneal with other sequences, but that are also homologous to E. coli sequences, and as such, may anneal with genomic DNA from E. coli).
3. Batch to Batch reproducibility
3B ULTRATOOLS, while being free of genomic DNA from E. coli, is of recombinant origin. This means that, in contrast to native polymerases, Batch to Batch reproducibility is guaranteed.
4. How much enzyme has to be used?
It depends on each experiment. For standard amplifications, 1 unit / 50 µl final reaction volume is enough. For long PCRs (products longer than 2 Kbp), PRINS, in situ PCR, or PCRs of more than 30 cycles, it may be necessary to add more enzyme (from 2 units up to 4 units / 50 µl final reaction volume).
5. Why two buffers?
Standard buffer has MgCl2 included in it, at a concentration of 2 mM. It is the usual buffer of choice for routine applications. However, if the experiment has to be optimised, we recommend to use Mg-free buffer, and optimise the MgCl2 accordingly.
6. Low yield
The first question that must always be made: has Mg concentration been optimised? If the concentration has been optimised, further investigation must be made on the experiment in order to know what has happened. Do not hesitate to contact us for further technical assistance, e-mails, phone calls, faxes .

Termi-DNA-Tor
1. Surfaces
Use of Termi-DNA-Tor is not recommended for surfaces sensitive to ethanol.
2. Automatic pipettes
Dip the autoclavable part into Termi-DNA-Tor. This does not affect the pipette. For non-autoclavable pipettes, dip the stick.
DNTPs
1. Storage
dNTPs are affected by freeze/thaw cycles. This affects them more than storage at 4 ºC or even room temperature.
2. Yields on amplification experiments
Adding more dNTPs will not necessarily result in better amplification yields. Except some experiments, where a slighly higher dNTP concentration is required (e.g. long amplifications), most experiments will have a decrease in yield when adding an excess of dNTPs (due to excess substrate), as well as lower copy fidelity.

 

Markers
1. Storage of markers
For frequent use, it is recommended to store markers at 4 ºC (up to 6 months), adding the corresponding loading buffer. Avoid repeated freeze/thaw cycles.
2. Quantitation
Some markers can be used for quantitation. In order to perform quantitation, gels must be stained with ethidium bromide after electrophoresis, and marker and sample volumes must be similar. Alternatively, quantitation can be performed with gels including ethidium bromide. However, electrophoresis buffer must also have the same ethidium bromide concentration (and this is not recommended, as ethidium bromide is a mutagenic agent).
A good estimation for gels including ethidium bromide (without ethidium bromide in the electrophoresis buffer) is to use the table attached to the marker, then multiply by 5-10 the result, in order to obtain an approximate value for DNA amount.
3. Marker not seen as in picture
Pictures depicting markers have been done with a given agarose concentration. The way markers are seen will depend on the agarose percentage.
4. Loading buffer
We prefer not to include the loading buffer in the markers, so that customer can choose (acridine orange, bromophenol blue, etc.).

Agaroses

Agaroses do not usually pose serious problems to customers. It is just a question of providing samples and them trying the product.
The only thing to note here is that melting of the agaroses requires boiling in TAE or TBE in a microwave oven or in a flame. After boiling, shake the flask vigorously, and heat for two more minutes.
Neither MB nor HR agarose are low melting point agaroses.
Biofood Identificacion
1. Detection limit
1 % is an average value, but this value will depend on the identity and degree of processing of the sample. We have obtained detection limits down to 0.5 % with some heterogeneous feed samples containing 3 species, but prefer to indicate 1 %, as this is a standard value for all samples.
2. Milk presence
BIOFOOD Identification detects presence of different animal species in a given sample. Milk, as well as serum, usually has cells remaining. This means that DNA is present, and will therefore be detected. A frequent question by customers in animal feed industry is whether a sample where milk or serum has been added will render a positive result. The answer depends on the treatment to which the milk or the serum has been subjected prior to addition to the sample. If it has been added raw, it will render a positive result. If it has been centrifuged or filtrated, there is the possibility that no cells remain. Please contact our Technical Dpt. for further information.
3. Fatty samples
For samples having a high content in fat, we recommend using an alternative protocol for DNA purification, in order to completely remove all fats prior to amplification, as this may inhibit the reaction.
In any case, raffinated oils are not suitable for analysis. As their manufacturing process involves filtration and centrifugation, no cells remain (and therefore, no DNA is present to be analysed).
4. Quantitation
BIOFOOD Identification is not intended for quantitation of results. Some estimations can be performed when running samples of known composition in parallel. Still, this is considered as a semi-quantitation, and has no statistical value. Please enquire our Technical Dpt.
5. Number of species
BIOFOOD Identification can detect vertebrate species in heterogeneous samples. When samples contain more than 3 species, restriction patterns may elapse, thus difficulting analysis, and even making compulsory use of dedicated software for band analysis.
Biofood Mixed
1. Detection limit
2 % is an average value, but this value will depend on the identity and degree of processing of the sample. We have obtained detection limits down to 0.5 % with some homogeneous feed samples, but prefer to indicate 2 %, as this is a standard value for all samples.
2. Milk presence
BIOFOOD Mixed detects presence of 6 different animal species in a given sample. Milk, as well as serum, usually has cells remaining. This means that DNA is present, and will therefore be detected. A frequent question by customers in animal feed industry is whether a sample where milk or serum has been added will render a positive result. The answer depends on the treatment to which the milk or the serum has been subjected prior to addition to the sample. If it has been added raw, it will render a positive result. If it has been centrifuged or filtrated, there is the possibility that no cells remain. Please contact our Technical Dpt. for further information.
3. Fatty samples
For samples having a high content in fat, we recommend using an alternative protocol for DNA purification, in order to completely remove all fats prior to amplification, as this may inhibit the reaction.
In any case, raffinated oils are not suitable for analysis. As their manufacturing process involves filtration and centrifugation, no cells remain (and therefore, no DNA is present to be analysed).
4. Quantitation
BIOFOOD Mixed is not intended for quantitation of results. Some estimations can be performed when running samples of known composition in parallel. Still, this is considered as a semi-quantitation, and has no statistical value. Please enquire our Technical Dpt.
5. Homogeneous samples
BIOFOOD Mixed is intended for use in homogeneous samples. By homogeneous, we define samples that have one animal species present, or at least, one animal species is majoritary. For samples having a majoritary species, but also presence of other species detected by the kit, both species will be detected by BIOFOOD Mixed, rendering two or more bands.

Biofood Standard
1. Detection limit
0.5 % is an average value, but this value will depend on the identity and degree of processing of the sample. We have obtained detection limits down to 0.05 % with some animal feed samples, but prefer to indicate 0.5 %, as this is a standard value for all samples.
2. Milk presence
BIOFOOD Standard detects presence of vertebrate material in a given sample. Milk, as well as serum, usually has cells remaining. This means that DNA is present, and will therefore be detected. A frequent question by customers in animal feed industry is whether a sample where milk or serum has been added will render a positive result. The answer depends on the treatment to which the milk or the serum has been subjected prior to addition to the sample. If it has been added raw, it will render a positive result. If it has been centrifuged or filtrated, there is the possibility that no cells remain. Please contact our Technical Dpt. for further information.
3. Fatty samples
For samples having a high content in fat, we recommend using an alternative protocol for DNA purification, in order to completely remove all fats prior to amplification, as this may inhibit the reaction.
In any case, raffinated oils are not suitable for analysis. As their manufacturing process involves filtration and centrifugation, no cells remain (and therefore, no DNA is present to be analysed).
4. Quantitation
BIOFOOD Standard is not intended for quantitation of results. Some estimations can be performed when running samples of known composition in parallel. Still, this is considered as a semi-quantitation, and has no statistical value. Please enquire our Technical Dpt.
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