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  • PreCR® Repair Mix

    Description

    The PreCR® Repair Mix is an enzyme cocktail formulated to repair damaged template DNA prior to its use in the polymerase chain reaction (PCR), microarrays or other DNA technologies. PreCR is active on a broad range of DNA damages, including those that block PCR (e.g. apurinic/apyrimidinic sites, thymine dimers, nicks and gaps) and those that are mutagenic (e.g. deaminated cytosine and 8-oxo-guanine). In addition, it will remove a variety of moieties from the 3´end of DNA leaving a hydroxyl group. The PreCR Repair Mix will not repair all damages that inhibit/interfere with PCR. For example, it will not repair 8-oxo-7,8-dihydro-2'deoxyadenosines or fragmented DNA. In fact, the ligase present in the mix is very active at sealing nicks in DNA but does not ligate blunt ends or nicks near a mismatch effectively. This limits the possibility of chimeric gene formation as a product of ligation. The PreCR Repair Mix can be used in conjunction with any thermophilic polymerase.

    DNA is susceptible to many types of damage resulting from exposure to a variety of chemical or environmental agents, manipulation or simply aging. The DNA Damage and PreCR Table  lists some possible DNA samples and the impact of the damage they may undergo.

    Note: The extent of damage caused by exposure to different reagents can vary, and its importance will depend on how the DNA is being used.


    Table 1:
    Types of DNA Damage
    Figure 1: Repair of different types of DNA damage with the PreCR Repair Mix.
    The gel shows trial amplifications from damaged DNA that was either not treated (-) or treated (+) with the PreCR Repair Mix. Type of DNA damage is shown. Note: heat treated DNA is incubated at 99°C for 3 minutes. Marker M is the 2-Log DNA Ladder (NEB #N3200 ).

    Highlights

    • Suitable for PCR reactions, microarrays or other DNA technologies
    • Easy-to-use protocols
    • Does not harm template

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    ThermoPol® Reaction Buffer-2010X
    BSA20 mg/ml
    L1 Primer Mix20 μM
    UV damaged Lambda DNA 0.5 ng/μl
    β-Nicotinamide adenine dinucleotide (NAD+)-20100X
    PreCR Repair Mix1X

    Advantages and Features

    Applications

    • Repair of DNA prior to its use as a template in PCR or other DNA technologies.

    Quality Control

    Quality Assurance Statement

    • The PreCR repair mix was tested for the ability to rescue successful amplification from UV damaged Lambda DNA (NEB #N3011). The mix was also tested for the ability to cleave oligonucleotide substrates containing thymine glycol, deoxyuracil, or 8-oxo-guanine in the absence of dNTPs. The absence of dNTPs prevents full repair and allows the presence of specific repair-targeting activities to be evaluated.

    Notes

    1. * PreCR Repair Mix components: Taq DNA Ligase, Endonuclease IV, Bst DNA Polymerase, Fpg, Uracil-DNA Glycosylase (UDG), T4 PDG (T4 Endonuclease V) and Endonuclease VIII.
    2. Reaction Conditions: DNA template, 100 µM dNTPs, 1X ThermoPol Reaction Buffer, 1X NAD+ and 1 µl PreCR Repair Mix in a total reaction volume of 50 µl. Incubate at 37°C.
    3. 1X NAD+ Solution: 0.5 mM NAD+.
    4. dNTPs are not included with the PreCR Repair Mix.

    References

    1. Diegoli TM, Farr M, Cromartie C, Coble MD, Bille TW. (2012). An optimized protocol for forensic application of the PreCR™ Repair Mix to multiplex STR amplification of UV-damaged DNA.. Forensic Sci Int Genet. Jul, 498-503. PubMedID: 22001155

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Will PreCR ligate my DNA fragments?
    2. How much DNA will PreCR repair?
    3. What are the liquid volumes of the PreCR Repair Mix Components?
    4. Will treating my DNA with the PreCR Repair Mix hurt my reaction?
    5. What is the sequence of the L1 primer mix?
    6. How is the damaged DNA that you test the PreCR Repair Mix against created? Can I buy this damaged DNA separately?
    7. Why does my control reaction not give a detectable amplicon?
    8. Can I buy any of the PreCR Repair Mix components separately?
    9. Will the PreCR Repair Mix blunt the ends of the DNA?
    10. Why don’t I see the expected band from my repaired template?
    11. Is my buffer compatible and which reaction should I use?
    12. Does the PreCr Repair Mix insert random nucleotides into the sequence that it repairs?
    13. Does the PreCR Repair Mix contain any contaminating human DNA? What are the quality controls that are used to test that?
    14. Does the PreCR Repair Mix remove covalent modifications from DNA bases, such as biotin or digoxigenin? Does it repair mismatches/ extra bases in DNA?
    15. If there are gaps and nicks remaining from a ligation reaction, will the PreCR Repair Mix repair all of these, so that the DNA will be suitable for microinjection into mice, for example?
    16. Can the PreCR Repair Mix be used for paraffin-embedded DNA?
    17. The repaired DNA will be used for an Nsp1 or Sty1 digestion followed by an adapter ligation, and PCR. Do you recommend cleanup of the PreCR Repair Mix reaction prior to this process?
    18. Can the PreCR Repair Mix repair damage in both single and double stranded DNA? Or, does it require a double stranded DNA as a template?
    19. Is the addition of dNTPs necessary for the PreCR Repair Mix to work properly?
    20. If I had a DNA template with mutation sites (ie. 8-oxoguanine or deaminated cytosines) that are directly adjacent to each other on opposite strands would treatment with PreCR™ Repair Mix cause a double strand nick/break?
    21. What gap lengths can be repaired with the PreCR Repair Mix?
    22. Does the PreCR Repair Mix work with less concentrated amounts of DNA (e.g. 500pg-1ng) than the amounts recommended?
    23. When doing bisulfite treatments of templates, the subsequent PCR reactions can pose a problem as the DNA seems to be very labile after the treatment. Can the PreCR Repair Mix improve these PCR results?
    24. Is it necessary to clean the PreCR reaction prior to carrying out quantitative PCR?
    25. What other components may be necessary that are not supplied with the PreCR Reaction Mix?
    26. When working with fragments, will the ends be ligated together by the PreCR Reaction Mix?
    27. Can T4 DNA ligase be used to ligate across an abasic site?
    28. How are abasic sites repaired by the PreCR Reaction Mix? Are new nucleotides put in and ligated? Is the existing nucleotide repaired?
    1. Sequential Reaction Protocol for PreCR Repair Mix
    2. Standard Reaction Protocol for PreCR Repair Mix
    3. Control Reaction Protocol for PreCR Repair Mix

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