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Riboprobe Primer Set

For PCR amplification of cDNAs cloned into the PCR-TRAP« system for riboprobe preparation.

This reaction creates a template for riboprobe synthesis by placing the T7 and SP6 promoter sites adjacent to the insert.

 Cat. No.
Riboprobe Primer Set
P406



ReversePrime Labeling Kit
For labeling cDNA probes for use in "Reverse Northern" blots.

GenHunter's ReversePrime Kit provides a complete system for labeling cDNA for use in "Reverse Northern" blots to conduct differential screening of positive clones generated by differential display. This kit is ideal for either dot blot or colony hybridization screening of differential display PCR products cloned into the PCR-TRAP« Cloning System (Cat. No. P404). This improvement significantly reduces the amount of labor required for screening by Northern Blots or Ribonuclease Protection Assays and provides high specificity. It accurately identifies positives from false positives and reduces the amount of RNA needed to conduct differential display screening.

 Cat. No.
ReversePrime cDNA Labeling Kit
R701
ReversePrime

References

  • Zhang, H. et al.: Differential screening of gene expression difference enriched by differential display. Nucleic Acid Res. 1996, 24:2454-2455. The ReversePrime cDNA Labeling Kit is also available with dNTP (-dATP) for use with a-[32P] dATP (Cat. # R701-A).


  • ReversePrime Graphic

    Reverse Northern dot blot screening of cDNA fragments

    cDNA fragments identified by differential display to be expressed only in tumor cells were reamplified, cloned into the PCR-TRAP« cloning vector, and dot-blotted onto duplicate nylon filters. RNA from normal and tumor cells were then labeled with 32P with the ReversePrime cDNA labeling kit and hybridized differentially to the duplicate filters. Note many positives were identified on the blot probed with labeled tumor cell RNA.



    Individual components for the ReversePrime Kit sold separately
    DescriptionCat. No.Volume
    dNTP (-dCTP) (500ÁM)S50380 ÁL
    T20 Primer (2 ÁM)T2060 ÁL
    5X RT BufferS401500 ÁL
    MMLV Reverse TranscriptaseS40240 ÁL
    dH20DH201.2 mL



    HotPrime DNA Labeling Kit
    For optimized labeling of differential display fragments with radioactively labeled dATP.

    The HotPrime DNA labeling kit features three unique improvements over the traditional "random priming" method which allow for radioactive labeling of DNA probes with at least 5-10X more specific radioactivity. It is designed to efficiently label cDNA probes isolated from differential display for Northern or Southern blot analysis as well as library screening. However, the HotPrime Kit can also label DNA probes for any other application to a similar high specific activity.

    The first improvement over traditional random-prime kits involves using random decamers instead of hexamers. This ensures more efficient priming of DNA probes of any sequence based on the observation that primers shorter than 9 bases are inefficient in initiating DNA synthesis (Williams et al., 1991, Nucleic Acids Res., 18:6531-6535).

    Second, because the cDNA fragments from differential display are relatively short (150-700 bp), the incorporation of anchored oligo-dT primers into the labeling buffer ensures the "full length" anti-sense cDNA probes to be labeled, which greatly increases the chance for signal detection on the Northern blot or library screening.

    Third, the use of radioactive dATP instead of dCTP allows labeling of DNA to the highest specific activity by taking the advantage of the AT rich nature of the 3' untranslated regions of mRNA detected by differential display.

    The HotPrime DNA Labeling Kit is also available with dNTP (-dCTP) for use with a-[32P] dCTP (Cat. # H501-C).


     Cat. No.
    HotPrime DNA Labeling Kit
    H501
    Components

    Graphic


    Northern blot analysis of differential expression of cDNA probe isolated by differential display.

    The band of interest from differential display was excised (A, arrowhead),reamplified and labeled with the Hotprime DNA labeling kit as a probe for Northern blot confirmation (B).



    Individual components for the HotPrime Kit sold separately
    DescriptionCat. No.Volume
    dNTP (-dATP) (500ÁM)S50470 ÁL
    Klenow DNA Polymerase (20 units)H60120 ÁL
    10X Labeling BufferH60260 ÁL
    Stop BufferH603150 ÁL
    Control DNAH60420 ÁL


     
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