Lab Tips

1. For first time users of the ligation kits (Cat. No. A100500, A100510, A100520, A100550, A100560, & A100570), we strongly recommend that the ligation control procedure be carried out in parallel with the ligation reaction. The ligation control procedure is described in the Control Section at the end of the product instructions.
   
   
2. When using the Ligation Kits (Cat. No. A100500, A100510, A100520, A100550, A100560, & A100570), your sequence-specific oligonucleotide must contain the following sequence: 5' T T T T T C G 3' at its 3' end in order to successfully ligate to the dendrimer. We strongly recommend that the oligo be HPLC or PAGE purified. The integrity of your oligonucleotide should be checked by gel electrophoresis to ensure proper size and homogeneity.
   
   
3. Handle membranes only when wearing gloves or with forceps to minimize background. Please follow the membrane manufacturer's recommendations for fixing your target to the membrane. Do not allow the membrane to dry out during the procedure. Nitrocellulose membranes are not recommended for the non-isotopic Biotin kits (Cat. No. A100210, A100510, & A100560) or Digoxigenin kits (Cat. No. A100220, A100520, &A100570) as these membranes will interfere with signal generation from dioxetane-based substrates (e.g., CDP-Star® ).
   
   
4. Hybridization and wash temperatures should be selected on the basis of the melting temperature (T_m) of your oligonucleotide. T_m calculations should be based upon the "nearest neighbor" method of Breslauer et al. in 50 mM salt (PNAS 83: 3746, 1986, or http://alces.med.umn.edu/rawtm.html at the Virtual Genome Center at the University of Minnesota) without including the 7 bases at the 3' end used to attach your oligo to the dendrimer (Ligation kits only). In general, we recommend that both the hybridization and wash temperatures be 10 to 15ºC below the T_m of your oligo. Regardless of the T_m of your oligo, hybridization temperatures should not exceed 65ºC, and wash temperatures should not exceed 55ºC. Also, note that the SDS in the hybridization buffer tends to precipitate out of solution at temperatures below 37ºC.
   
   
5. Hybridization solutions which include high molecular weight polymers (e.g., Denhardt's Solution, dextran sulfate, or salmon sperm DNA) are not recommended for use with 3DNA dendrimers because they will decrease the signal. For optimal results, please prepare and use the hybridization solution specified in the product instructions.
   
   
6. The blocking reagent supplied with the kits is specific for the 3DNA dendrimer. Other blocking reagents should not be used as they may decrease the signal from the target sequence.