Sam's Notebook » hexokinase http://onsnetwork.org/kubu4 University of Washington - Fishery Sciences - Roberts Lab Thu, 08 Nov 2018 21:47:12 +0000 en-US hourly 1 http://wordpress.org/?v=4.0 PCR – Hexokinase Partial CDS http://onsnetwork.org/kubu4/2013/04/09/pcr-hexokinase-partial-cds/ http://onsnetwork.org/kubu4/2013/04/09/pcr-hexokinase-partial-cds/#comments Tue, 09 Apr 2013 22:15:24 +0000 http://onsnetwork.org/kubu4/?p=194

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…

]]> http://onsnetwork.org/kubu4/2013/04/09/pcr-hexokinase-partial-cds/feed/ 0 PCR – Hexokinase and Partial Exon #1 http://onsnetwork.org/kubu4/2013/04/08/pcr-hexokinase-and-partial-exon-1/ http://onsnetwork.org/kubu4/2013/04/08/pcr-hexokinase-and-partial-exon-1/#comments Mon, 08 Apr 2013 22:17:12 +0000 http://onsnetwork.org/kubu4/?p=196

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.

]]> http://onsnetwork.org/kubu4/2013/04/08/pcr-hexokinase-and-partial-exon-1/feed/ 0 PCR – Hexokinase Promoter and CDS (repeat from 20130227) http://onsnetwork.org/kubu4/2013/03/14/pcr-hexokinase-promoter-and-cds-repeat-from-20130227/ http://onsnetwork.org/kubu4/2013/03/14/pcr-hexokinase-promoter-and-cds-repeat-from-20130227/#comments Thu, 14 Mar 2013 22:20:18 +0000 http://onsnetwork.org/kubu4/?p=198

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!

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