Still have insufficient quantities of DNA for sequencing. Master mix calcs and cycling params are here. Additionally, used some of the purified PCR product as template in one of the reactions, just for comparison purposes. cDNA template was pooled cDNA from 20110311 from various C.gigas tissues. Also, increased the amount of template 4-fold in an attempt to obtain higher yields of PCR products for sequencing.
Results:
Lane 1: Hyperladder I (Bioline)
Lane 2: PCR 1 (cDNA template)
Lane 3: PCR 2 (cDNA template)
Lane 4: PCR 3 (PCR template)
Lane 5: Neg. Control
Bands were excised and will be purified using Ultra-free DA columns (Millipore). Also, it’s very clear that using the purified PCR product as template produced a much greater yield, although there appear to be some spurious, high-molecular weight banding/smearing.