Performed PCR using the primers CG_HK_CDS_2132-2158 (SRID: 1521) and Cg_Hk_CDS_3’_no_stop (SRID: 1519) on pooled C.gigas cDNA (from DATE).
Master mix calcs and cycling params are here.
Samples were run in duplicate.
Results:
No amplification of any kind. Time for some troubleshooting…
Performed PCR using newly designed primers to amplify the C. gigas hexokinase “promoter” (-2059bp from start) along with a portion of the first exon.
Primers used were Cg_Hk_Prom_pBAD_-2059 (SRID: 1518) and Cg_HK_Exon1_R (SRID: 1520).
Template used was C.gigas gDNA BB15 (from 20090519; 0.4216ug/uL). Master mix calcs are here. Cycling params are the same used on 20130227.
Samples were run in duplicate.
Results:
Lane 1: Hyperladder II (Bioline)
Lanes 2-3: C.gigas gDNA
Lanes 4-5: NTCs
We see a band of >2000bp (that’s the maximum on the molecular weight marker). The bands from each replicate were excised, purified using Ultrafree-DA columns (Millipore) and stored at 4C.
Performed a repeat of the failed PCR from 20130227, but used a pool of cDNA (made from 20110311 C.gigas cDNA) instead of a single sample and changed the annealing temp to 50C.
Results:
Same exact results as 20130227; nothing. As such, didn’t take gel image. Will retry one more time using a long-distance polymerase, along with varying [MgCl2].
UPDATE 20130318: Doh! When talking about this at lab meeting today, I realized I’m trying to amplify the promoter (a genomic sequence) using cDNA! Will re-design primers and develop new cloning strategy for this!