PCR - Abalone gDNA/RNA/cDNA w/new TOLLIP primer

Performed a new PCR on the three types of samples listed above with new TOLLIP primers. The new TOLLIP primers (H.discus_806_F/Ab_866_Rv) surround a putative intron. Thus, they will be useful for determining the presence of gDNA contamination in RNA/cDNA. Anneal temp 55C. PCR set up is here .

Lane 1 - Hyperladder

Lane 2 - gDNA

Lane 3 - cDNA (QT)

Lane 4 - RNA (untreated)

Lane 5 - DNased RNA

Lane 6 - QT Kit, No RT

Lane 7 - H2O

Lane 8 - H2O

Results: This could possibly be the most confusing gel I’ve ever had the “pleasure” of running/analyzing, despite that it only has 7 samples.

No band in the gDNA sample, which could be explained by the intron size being too large for amplification with a basic polymerase. The cDNA worked as expected and contains a band of ~150bp. The RNA sample has a faint band of ~750bp. The DNased RNA sample has a band of ~400bp. The differences seen betweeen the gDNA (Lane 2), RNA (Lane 4) and DNased RNA (Lane 5) are truly bizarre. The No RT sample has no band. And, to top things off, one of the H2O samples is blank , but the other one has a band of ~1500bp! Ugh. How is all of this even possible?