Step 8. PCR amplification with Tag specific primer (Ptag)
At this stage, you could try separate amplifications with different Ptag/M13F primer pairs to obtain specific PCR product for each of the novel tags using 1ul of 1/50 and 1/300 (dilution) amplified rSAGE library as template. Hot-start and touchdown PCR is highly recommended. However, a separate step of Hot-start is not necessary if Platinum Tag DNA polymerase (GibcoBRL Cat. No. 10966018) is used with an initial cycle of 2 minutes at 94oC as described below. PCR conditions might require further optimization in terms of cycle number, template dilutions (amplified rSAGE) and temperature.
Step 9. PCR product isolation and sequencing
After visualizing specific PCR product on an agarose gel, individual bands are excised and purified. We currently use Qiaquick gel extraction kit (Qiagen, Cat. No.28704)
Sequencing purified PCR product can be performed by standard dideoxynucleotide termination method either manually or by automatic sequencer. M13F and Ptag both can be used as sequencing primers.
Step 10. Verify Tag sequence
Once complete sequence of the PCR product is obtained, it’s recommended to check whether the correct tag is present in the PCR product or it is indeed after the last CATG before PolyA signal, if possible, before further analysis.
If the presence of the tag is confirmed, the additional nucleotide sequence obtained, usually hundreds of base pair long, can help blast search considerably. If you are still unable match it to known sequence or ESTs, you might have a novel gene.(Congratulation!) The purified PCR product can be used as probe on northern blot analysis, and library screening. The additional sequence can also help you design primers for 5’ RACE to isolate full length cDNA if the mRNA is small.
Reagents
Please see SAGE protocol for solutions, Dynal beads and commonly used methods. It’s highly recommended to follow all the specifications for the reagents in SAGE protocol unless specified otherwise in rSAGE protocol.
Primers and Linkers
