Cod UNG FAQ

  1. Can I replace E. coli UDG with Cod UNG™ in all applications?
  2. What is the advantage of heat labile Cod UNG™ over non heat labile UDGs?
  3. What might be the disadvantage of the heat lability of Cod UNG
  4. What is the activity of Cod UNG™ in Taq buffer with 1.5 mM – 4 mM of MgCl2?
  5. What is the activity of Cod UNG™ in common T4 DNA Ligase buffers?
  6. What is the optimal reaction temperature of Cod UNG™?
  7. What salt concentration is optimal for Cod UNG™?
  8. What pH is optimal for Cod UNG™?
  9. What is the recommended Cod UNG™ amount per reaction?
  10. What is the molecular weight of Cod UNG™?
  11. Is Cod UNG™ a tagged protein?
  12. Does the Cod UNG™ preparation contain BSA?
  13. What is the inactivation temperature of Cod UNG™?
  14. How stable is Cod UNG™?
  15. Does Uracil Glycosylase inhibitor (UGI) inhibit Cod UNG™?
  16. Does Cod UNG™ remove uracil from both ss and ds DNA with the same efficiency?
  17. Does Cod UNG™ remove ribouracil from RNA?
  18. Can Cod UNG™ be used to remove dU from a short 25mer oligo?
  19. What is the difference between UDG and UNG?
  20. Is Cod UNG™ eliminating carry over contamination in PCR at 100%?
  21. Do I need to store my PCR product immediately after the PCR at -20˚C or to re-purify it asap when Cod UNG™ was used?
  22. Is the dU containing DNA suitable for electrophoresis?
  23. Can I clone dU containing DNA?
  24. Can I digest dU containing DNA with restriction enzymes?
  25. Is it possible to use dU containing DNA for protein-DNA interaction studies?
  26. Can I use dU containing DNA for sequencing?
  27. Can I use the dU containing DNA as a probe for hybridization?
  28. How can I visually check if my Cod UNG™ is really active and working?
  29. I used UNG in my PCR, but I still have a band in my negative control without the template. How could this happen?

1. Can I replace E. coli UDG with Cod UNG™ in all applications?

Cod UNG™ can be used in all applications where other UDGs are used, unless UNG activity is required at temperatures above 40˚C.

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2. What is the advantage of heat labile Cod UNG™ over non heat labile UDGs?

There are three main advantages of using heat labile Cod UNG™:

A. Cod UNG is completely and irreversibly in activated. Unlike other UDGs, the PCR products obtained using dUTP and Cod UNG ™ are not degraded after the PCR and are therefore suitable for cloning, restriction digestion, sequencing and other applications.

B. Cod UNG ™ enables contamination control in RT-PCR, qRT-PCR and One step RT-PCR. This is not possible when using other UDGs since they are active simultaneously as reverse transcriptase, thereby digesting newly synthesized cDNA. Cod UNG ™ is inactivated at temperatures above 45˚C, ensuring that cDNA containing dUTPs are left intact.

C. The total reaction time and temperature can be reduced. Other UDGs typically introduces a 10 minutes incubation step at 37 – 40˚C. Cod UNG ™ is active at room temperature, and carry over prevention is completed within 5 minutes after mixing all components.

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3. What might be the disadvantage of the heat lability of Cod UNG

Cod UNG™ is not suitable for applications where the enzyme activity at higher temperatures is required (45˚C or above), or if any pre-heating above 45˚C are necessary after Cod UNG ™ is added to the reaction mix.

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4. What is the activity of Cod UNG™ in Taq buffer with 1.5 mM – 4 mM of MgCl2?

Cod UNG™ is active in all common PCR, RT-PCR qPCR buffers and Master mixes tested in our laboratory.
The activity of Cod UNG™ in common Taq buffers with 1.5 mM – 4 mM MgCl2 is close to 100%.

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5. What is the activity of Cod UNG™ in common T4 DNA Ligase buffers?

The activity of Cod UNG™ in common ligation buffers is close to 100%.

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6. What is the optimal reaction temperature of Cod UNG™?

The optimal temperature for Cod UNG™ activity is 37°C. Effective temperature range is +20 to +40˚C.

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7. What salt concentration is optimal for Cod UNG™?

The optimal salt concentration for Cod UNG™ is 50 mM NaCl. Increasing salt decreases the activity of Cod UNG.

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8. What pH is optimal for Cod UNG™?

The optimal pH for Cod UNG™ is pH 7.5. Cod UNG is active between pH 7 and 9.

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9. What is the recommended Cod UNG™ amount per reaction?

The optimal amount of Cod UNG™ is typically 0.1-1 U per 50 μl reaction volume.

However, the optimal amount of enzyme is depending on the particular application and we would recommend determining it empirically.

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10. What is the molecular weight of Cod UNG™?

The Molecular Weight of Cod UNG™ is 28 kDa.

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11. Is Cod UNG™ a tagged protein?

No, Cod UNG™ is not a tagged protein.

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12. Does the Cod UNG™ preparation contain BSA?

No, BSA is not used in the storage buffer of the Cod UNG™.

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13. What is the inactivation temperature of Cod UNG™?

Cod UNG is quickly inactivated at temperatures of 50°C and above. Complete and irreversible inactivation of Cod UNG™ is achieved when incubating at 55˚C for a minimum of 20 minutes, or at 95˚C for 1 sec (inactivation is complete during ramping).

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14. How stable is Cod UNG™?

We guarantee the stability of Cod UNG™ for 24 months if stored in the original vial at -20˚C.

We guarantee the stability of Cod UNG™ for 6 months if stored in the original vial at +4˚C.

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15. Does Uracil Glycosylase inhibitor (UGI) inhibit Cod UNG™?

Yes. As other UNGs, Cod UNG binds UGI in a 1:1 ratio. However, Cod UNG™ does not require any inhibitors as it is completely and irreversibly inactivated when incubated at 55˚C for 20 minutes.

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16. Does Cod UNG™ remove uracil from both ss and ds DNA with the same efficiency?

No, Cod UNG™ removes uracil from ssDNA in a rate of 200% compared to dsDNA.

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17. Does Cod UNG™ remove uracil from RNA?

No, Cod UNG™ will not act on uracil on ribose sugar backbone (ribouridine).

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18. Can Cod UNG™ be used to remove dU from a short 25mer oligo?

Yes, Cod UNG™ will act on 25 mer oligos containing Uracil. The minimal substrate for Cod UNG is <5 mer oligos.

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19. What is the difference between UDG and UNG?

Originally, the ung is the name of E. coli gene coding the Uracil DNA Glycosylase, and UDG is the name of the protein.

However nowadays some suppliers use the UDG abbreviation for Uracil DNA Glycosylase, others use the UNG abbreviation which applies to another common name of the enzyme, Uracil-N-Glycosylase. Both abbreviations are common and correct.

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20. Is Cod UNG™ eliminating carry over contamination in PCR at 100%?

If used as recommended, the Cod UNG™ will degrade all Uracil containing DNA from previous PCR. Only the new template which does not contain Uracil will be amplified. But decontaminating very small amplicons containing few Uracils can be less efficient.

However, one should not forget to take typical measures against PCR contamination, as the PCR can be contaminated at any time with new templates without Uracil which are not substrates for UNG.

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21. Do I need to store my PCR product immediately after the PCR at -20˚C or to re-purify it asap when Cod UNG™ was used?

No, Cod UNG™ is fully inactive after a PCR, ensuring that no degradation of Uracil containing PCR products occurs. Other common UDGs used for PCR carry-over contamination prevention reactivate at moderate temperatures and degrade the dU containing PCR products. This is not the case when working with Cod UNG™.

This unique feature of Cod UNG™ allows to use the PCR product directly for cloning or sequencing and to store it without any special time or temperature limitations.

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22. Is the dU containing DNA suitable for electrophoresis?

In gel electrophoresis, dU containing DNA performs as well as usual DNA. It migrates in the same way and is stained with same efficiency as dT containing DNA.

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23. Can I clone dU containing DNA?

Yes, one can use dU containing DNA for cloning just as well as usual dT containing DNA.

For successful restriction digests, please check the properties of the chosen restriction enzyme (for example EcoRI and BamHIdigest dU containing DNA well, whereas HpaI, HindII, HindIII show reduced activity on these substrates).

For successful cloning, dU containing DNA should be transformed into ung– bacterial hosts.

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24. Can I digest dU containing DNA with restriction enzymes?

Yes, one can use dU containing DNA for restriction digestions, but there are some limitations due to the specificities of certain restriction enzymes.

For successful restriction digests, please check the properties of the chosen restriction enzyme (for example EcoRI and BamHIdigest dU containing DNA well, whereas HpaI, HindII, HindIII show reduced activity on these substrates).

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25. Is it possible to use dU containing DNA for protein-DNA interaction studies?

dU-containing DNA may interfere with protein binding or DNA-protein interaction studies.

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26. Can I use dU containing DNA for sequencing?

dU containing DNA performs as well as usual DNA with dT in dideoxy sequencing.

It has been proved experimentally that the dU containing PCR product gives excellent sequencing results, after use of Cod UNG™ for carry-over contamination prevention. With other UNGs the dU containing PCR product is degraded during the storage due to the reactivated enzyme and therefore sequencing becomes unreliable.

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27. Can I use the dU containing DNA as a probe for hybridization?

dU containing DNA performs as well as usual DNA with dT in hybridization.

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28. How can I visually check if my Cod UNG™ is really active and working?

Incubate the dU containing ds DNA with Cod UNG™ for 5 min at room temperature in 1x Cod UNG™ Reaction Buffer, heat and visualize on the agarose gel. The DNA should be degraded compared to untreated control.

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29. I used UNG in my PCR, but I still have a band in my negative control without the template. How could this happen?

If used as recommended, the Cod UNG™ will degrade all Uracil containing DNA from previous PCR. Only the new template which does not contain Uracil will be amplified.

However, one should not forget to take typical measures against PCR contamination, as the PCR can be contaminated at any time with new templates without Uracil which are not substrates for UNG.

Foreign template contamination can result from contaminated reagents, pipettes, water and so on. The PCR decontamination kit from ArcticZymes can be used to reduce contamination from non-dUTP-containing sources.

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