Direct sequencing of PCR products

Direct sequencing of PCR products generally gives very good results, provided that the initial PCR reaction yields only one product or one product can be gel purified.

1. Run agarose gel to check whether the PCR reaction yields more than one product.

2. If only one product is visible, primers and dNTPs can be cleaned up using Qiaquick spin column, SAP/ExoI, or Micorcon.

3. If there are multiple bands, gel purification is required.

4. Quantitate the purified PCR product. Use 10 ng DNA per 100 base to be sequenced.

5. Sequencing by cycle sequencing with the original primers or internal primers.