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5'/3' RACE Kit, 2nd Generation Troubleshooting

Product No. 03353621001

Troubleshooting

POOR RESULTS

In addition to the troubleshooting provided in the product manual, most probably the efficiency of the tailing reaction performed by Terminal Transferase could be impaired. This could occur due to several reasons (which will not only affect the control reaction, but 5′ RACE in general):

  • Improper denaturation
    Check if the denaturation step prior to the tailing reaction was performed properly according the protocol: Incubate for 3 min at +94 °C. Chill on ice.
  • Insufficient incubation time
    Note that the incubation period for the tailing reaction can be increased to 30 min.
  • Incorrect storage of the High Pure PCR Product Purification Kit
    In this case, precipitation within the Binding Buffer may occur. Such precipitates of buffer components can be carried over to the final eluate and inhibit the subsequent Terminal Transferase reaction.
  • cDNA purification protocol
    Strictly follow the purification protocol in the product manual of the 5’/3’ RACE.
    In particular, it is important that the centrifugation step after the last washing step (prior to elution) is performed at maximum speed of 13,000 x g for a minimum of 2 min. The filter tube should be completely dry before elution of bound cDNA. Otherwise, residual ethanol inhibits the tailing reaction. All other centrifugation steps should be performed at 6,000 to 8,000 x g, and for these a 30s duration is sufficient.


Stretches of 60 to 100 T bases in the 5’ region

When sequencing the final PCR product of the 5´-RACE procedure one can detect stretches of 60 to 100 T bases in the 5´region. Normally the presence of a non-T base at the 3´-end of the PCR-anchor primer prevents this from occurring by forcing the primer to bind to the inner end of the poly(A) tail.
Nevertheless there are several reasons that T-base stretches may occur:

  1. If the annealing conditions during the first PCR amplification are not optimal (i.e., the oligo-d(T)-anchor primer is not completely annealed and might bind within the 5´poly(A) tail) and a proofreading polymerase (e.g., Expand High Fidelity PCR System or Expand Long Template PCR System) is used instead of Taq DNA polymerase, the 3´->5´exonuclease activity may remove a non-annealed 3´ non-T anchor base. This can lead to priming of PCR from within the poly-A tail and the introduction of long 5´ T stretches into the PCR product. Stringent annealing conditions are very important for success of this reaction.
  2. Correct purification of the first strand cDNA is extremely important to remove residual unincorporated nucleotides. Otherwise in the 5´tailing reaction, other nucleotides than A can be incorporated which leading to internal binding of the oligo-d(T)-anchor primer to the 5´poly(A) tail.

Materials

     

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