20130409
PCR - Hexokinase Partial CDS
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...
20130408
PCR - Hexokinase and Partial Exon #1
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.
20130314
PCR - Hexokinase Promoter and CDS (repeat from 20130227)
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!
20130227
PCR - Hexokinase Promoter and CDS
Performed PCR to amplify the C.gigas hexokinase (ACCESSION#) promoter region (-2059bp) and the CDS without the stop codon. Elimination of the stop codon allows for subsequent cloning into the pBAD-TOPO expression vector, which will incorporate the V5 epitope tag sequence. This tag will be used to distinguish between endogenous hexokinase expression and expression generated from our hexokinase construct.
Used hexokinase primers (SR IDs: 1518, 1519).
Master mix calcs and cycling params are here.
Results:
Ladder: Hyperladder II (Bioline)
No amplification in sample or no-template control. Will re-do with lower annealing temp (50C).
20130213
Reverse Transcription - Herring RNA from 20091026
Performed an RT reaction on pooled herring gonad and liver mRNA from
20091026 for James Raymond at the UNLV. A single RT reaction was performed using 12.75uL (208ng) of the pooled gonad mRNA and 5uL (132.5ng) of the pooled liver RNA, according to our default MMLV (Promega) protocol. After reaction was completed, sample was stored @ -20C and then shipped to James Raymond on 20130214.