Samples from yesterday were centrifuged 30mins, 4000g, 4C (fixed angle rotor).
Supernatant was removed and pellets were dried (inverted tubes for 30mins).
Pellets were resuspended in 200uL H2O.
Data spreadsheet is here: 20131125 – Oyster Glycogen Assay
Finally located the remaining half of Emma’s samples. These had already been freeze dried AND pulverized! So, I just had to weigh out ~half of each sample for the glycogen assay.
Glycogen Assay:
Samples were placed in 3mL of 15% trichloracetic acid (TCA), vortexed and incubated for 1hr @ 4C.
Samples were spun 10mins, 3,000g, at 4C (in bucket rotor).
500uL of supernatant was transferred to 4mL of 100% EtOH, vortexed and incubated O/N @ 4C.
Will continue processing tomorrow.
Data spreadsheet is here: 20131125 – Oyster Glycogen Assay
Samples from yesterday were centrifuged 30mins, 4000g, 4C (fixed angle rotor).
Supernatant was removed and pellets were dried (inverted tubes for 30mins).
Pellets were resuspended in 200uL H2O and stored @ -20C.
Data spreadsheet is here: 20131125 – Oyster Glycogen Assay
Samples were previously freeze dried overnight and stored @ -20C. To maximize sample homogeneity and, thus, increase accuracy of both assays, all samples were mechanically pulverized in their existing tubes. Approximately half of each sample was weighed and used for the glycogen assay. The remainder of each sample was stored @ -20C.
Glycogen Assay:
Samples were placed in 3mL of 15% trichloracetic acid (TCA), vortexed and incubated for 1hr @ 4C.
Samples were spun 10mins, 3,000g, at 4C (in bucket rotor).
500uL of supernatant was transferred to 4mL of 100% EtOH, vortexed and incubated O/N @ 4C.
Will continue processing tomorrow.
Data spreadsheet is here: 20131125 – Oyster Glycogen Assay
Repeated PCR from 20130919, but with a newly designed reverse primer. Primers were designed using MethPrimer (SR IDs: 1553, 1555). Primers were designed using MethPrimer:
Results:
No amplification of any samples BS-treated or non-treated.
Repeated PCR from 20130919, but with a newly designed set of primers that targets the desired region of C1q for sequencing (SR IDs: 1553, 1554). Primers were designed in Geneious.
Results:
No amplification of any samples BS-treated or non-treated.
Ran PCR on lake trout DNA and lake trout bisulfite-converted DNA. Used primers SRIDs: 1551 and 1552. DNA was isolated by Caroline Storer on 4/4/2011 and bisulfite converted on 4/7/2011. Master mix and cycling params are here:
http://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20130919-01.jpg
Samples:
Lake Trout Lean_6 Liver
Lake Trout Lean_7 Liver
Lake Trout Siscowet_6 Liver
Lake Trout Siscowet_7 Liver
Bisulfite converted DNA from the four samples listed above.
Results:
Lane 1: Hyperladder II (Bioline)
Lane 2: Lean6
Lane 3: Lean6 BS
Lane 4: Lean7
Lane 5: Lean7 BS
Lane 6: Siscowet6
Lane 7: Siscowet6 BS
Lane 8: Siscowet7
Lane 9: Siscowet7 BS
All the non-BS converted samples amplified as expected, producing a band of ~560bp. However, none of the BS-converted DNA produced any amplification. It is likely an issue with the primer sequences and the resulting conversion of the gDNA.
Will look at Caroline Storer’s notebook entries for her work on this and try to evaluate what has already been done.
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.
