Tag Archives: gel

DNA Isolation – Test Sample

Due to the recent poor quality gDNA that has been isolated from C.gigas gonad, I decided to do a quick test using TE for DNA pellet resuspension in hopes that old Buffer EB (Qiagen) or old nuclease-free H2O (Promega) are to blame for the apparent, rapid degradation that I’ve experienced.

Isolated gDNA from a C.gigas female gonad sample (EV2 141 go) provided by Mac. Isolated gDNA using DNazol (Molecular Research Center):

  1. Incubated ~25mg of tissue O/N @ RT in 500uL of DNazol + 100ug/mL Proteniase K (2.7uL of 18.5mg/mL Fermentas stock) on rotator.

  2. Added additional 500uL of DNazol and briefly disrupted remaining tissue with a few pipette strokes.

  3. Pelleted debris by spinning 10mins, 10,000g @ RT.

  4. Transferred supe to new tube and repeated Steps 3 & 4 one time.

  5. Added 500uL of 100% EtOH; mixed by inversion.

NOTE: Despite initial appearance of white cloudy appearance after EtOH addition, cloudiness dispersed upon inversion and no visible DNA strands were present

  1. Pelleted DNA by spinning 5000g 5mins @ RT.

  2. Removed supe and washed pellet with 1mL of a 70% DNazol+30% EtOH solution.

  3. Removed supe and washed pellet with 1mL 70% EtOH.

  4. Repeated Step 8 two times.

  5. Discarded supe, quick spun tube to pool residual EtOH. Removed all residual EtOH.

  6. Resuspended in 200uL of TE (pH = 8.0) and incubated at RT for 5mins.

  7. Pelleted insoluble material 12,000g 10mins @ RT.

  8. Transferred supe to clean tube.

  9. Spec’d on NanoDrop1000.

  10. Ran ~500ng on 1.0% agaroase 1x modified TAE gel to evaluate integrity.

Results:

260/280 value looks excellent, but, as always seems to be the case with DNazol/TriReagent, the 260/230 value looks crappy. Will investigate gDNA integrity on agarose gel.

Gel Loading:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – EV2 141 go C.gigas female gonad gDNA

Well, look at that! A nice, clear, high molecular weight band! It looks like my Buffer EB and/or nuclease-free water are is contaminated. Have discarded both. Will re-isolated Claire and Mac’s gDNA.

Phenol-Chloroform DNA Clean Up – Mac and Claire’s Samples (from 20140410)

Due to low 260/230 values and Mac’s smeary sample, performed a phenol-chloroform DNA cleanup on the samples isolated 20140410.

  1. Brought volume of each sample to 200uL with Buffer EB (Qiagen).

  2. Added an equal volume (200uL) of 25:24:1 Phenol/Chloroform:Isoamyl alcohol.

  3. Mixed on rotator for 20mins @ RT.

  4. Separated aqueous/organic phases by spinning at 12,000g 5mins @ RT.

  5. Transferred aqueous phase to new tube. Repeated steps 2-4 until samples exhibited no more interphase. Combined aqueous phases in to a single tube for each of the two samples.

  6. Added and equal volume of chloroform (170uL).

  7. Mixed on rotator for 20mins @ RT.

  8. Separated aqueous/organic phases by spinning at 12,000g 5mins @ RT.

  9. Transferred aqueous phase to new tube.

Performed an ethanol precipitation on each sample.

  1. Added 0.1 volumes of 5M sodium acetate (pH = 5.2).

  2. Added 2 volumes of ice cold 100% EtOH.

  3. Incubated 20mins @ -20C.

  4. Pelleted DNA by spinning 16,000g, 20mins @ 4C.

  5. Discarded supe and washed pellets with 1mL 70% EtOH.

  6. Pelleted DNA by spinning 16,000g, 5mins @ 4C.

  7. Repeated steps 5-6 one time.

  8. Removed all supernatant and resuspended in 100uL of nuclease-free H2O.

  9. Spec’d on NanoDrop1000.

NOTE: Mac’s sample exhibited the same chunky/cloudiness upon addition of 100% EtOH that has been seen previously by both her and myself…

Results:

So, the clean up seemed to work wonders on the 260/230 values. Not surprisingly, Mac’s sample didn’t clean up nearly as nicely as Claire’s, based on my observations of the odd behavior during EtOH precipitation.

And, despite the nice, clean looking peaks, the 260/280 ratios are actually WORSE than the original isolation. Will run on gel for a further assessment of quality/integrity.

Loaded 5uL of each sample (~600ng) on a 1.0% agarose, 1x modified TAE gel stained with ethidium bromide.

Gel Layout:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – Claire’s CgF gonad sample

Lane 3 – Mac’s gonad sample

Used Hyperladder I this time, which has a high molecular weight band of 10kb and a low molecular weight band of 200bp.

Well, this totally sucks. Both samples appear to consist of nothing but 150-200bp fragments. Is something actually degrading these samples? The Buffer EB I used during the initial extraction is certainly old. Possible source of degradation? Ugh. Maybe I’ll try this again, but resuspend in TE…

DNA Isolation – Claire’s C.gigas Female Gonad and Mac’s C.gigas Gonad

Due to the poor quality DNA yielded by the DNeasy Kit (Qiagen; see 20140404), I am re-isolating these samples using DNazol (Molecular Research Center). Weighed tissue from each frozen sample:

Claire’s (Female DNA; 5/6/2013) – 0.022g

Mac’s (EV2 9.g) – 0.017g

Incubated samples in 500uL of DNazol + 100ug/mL Proteinase K (2.7uL of 18.5mg/mL stock) O/N at RT on rotator. An additional 500uL of DNazol was added, mixed by pipetting to break up remaining tissues clumps. Manufacturer’s protocol was followed, substituting the first EtOH wash with a wash of 70% DNazol, 30% 100% EtOH. Samples were resuspended in 100uL Buffer EB (Qiagen) and spec’d on a NanoDrop1000.

NOTE: Mac’s sample seemed to get “chunky”/cloudy during the precipitation portion of the procedure. Claire’s remained clear. Although not noted, Mac’s sample behaved in a similar fashion when adding Buffer AL to the sample when using the Qiagen DNeasy Blood & Tissue Kit. Finally, Mac has previously mentioned this behavior to me as well.

Results:

Suprisingly high yields from Mac’s sample.

Both samples exhibit poor 260/230 ratios and high absorbance at 230nm is evident in both samples. Mac’s sample may benefit from

Ran ~600ng of each sample on a 0.8% 1x modified TAE agarose gel to visually assess sample quality.

Gel Loading (from left to right):

  1. Hyperladder II (Bioline)

  2. Claire’s Female DNA

  3. Mac’s gonad (EV2 9.go)

I knew the ladder was of little use due to high molecular weight of gDNA, but it still serves as a bit of a reference. Highest molecular weight band is 2000bp.

Claire’s sample looks pretty good, in relation to the lack of smearing. A single, high molecular weight band is present (albeit, faint) with almost no smearing. However, I’m disappointed by the lack of definition in the band. I fully expected a sharper, more defined band.

Mac’s sample shows a high molecular weight band and significant smearing. Smearing could be indicative of either DNA degradation or high amounts of RNA carryover. If the latter, could explain the high yield.

Will attempt to clean up both samples (RNase and/or do a chloroform clean up).

DNA gel – Claire’s C.gigas Female Gonad and Mac’s C.gigas Gonad

Ran out 2uL of Clair’es C.gigas female gonad gDNA (from 20140328) and Mac’s C.gigas gonad gDNA (from 20140402) for quality assessment. Both samples had been isolated using Qiagen’s Blood & Tissue DNeasy Kit. 2uL of each sample was run on a 0.8% 1x TBE gel.

Results:

Loading:

Lane 1 – Hyperladder 1 (Bioline)

Lane 2 – Claire’s gDNA

Lane 3 – mac’s gDNA

Both samples show an extremely high amount of smearing. Additionally, both samples have definitive bands that correspond to ~1300bp and ~850bp.

DNA Quality Check – Yanouk’s Oyster gDNA

We’ve had some Illumina sequencing issues with Yanouk’s samples, so I ran the samples out on a 0.8% agarose gel to evaluate the levels of degradation. Loaded 2uL of each sample. Did not load equal quantities of gDNA, due to the lack of available gDNA in the samples we submitted for Illumina sequencing. Added 2uL of H2O to samples 37 & 38 in hopes of having sufficient DNA for visualization on the gel.

See Sam’s Notebook 20131004 for sample concentrations.

Results:

First lane is Hyperladder II (Bioline). Highest molecular weight of the ladder is 2kb.

Sample numbers are listed above each lane.

All samples show a significant amount of smearing, but all still have an identifiable high molecular weight band. Will show to Steven and discuss options for re-sequencing.

PCR – Lake Trout C1q

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.

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.

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).

PCR – COX/PGS Cloning Colony Screens from yesterday

Performed PCR on 40 colonies using both qPCR primer sets to see if I could differentiate between which colonies potentially contained each isoform to reduce the amount of clones needed for sequencing. Master mix and cycling params are here. Primers used were:

Cg_COX1/2_qPCR_F (SR ID: 1192)

Cg_COX1_qPCR_R (SR ID: 1191)

Cg_COX2_454align1_R (SR ID: 1190)

Positive controls for both primers set were also run. The positive control template was the purified PCR product from 20111006.

Results:

Ladder is Hyperladder II (Bioline). Samples are loaded, left to right, as PGS1 and PGS2 on each colony (e.g. on the bottom gel image, under the “Colony 40″ label is the PGS1 rxn on the left and the PGS2 rxn on the right).

Nearly every colony exhibits amplification using both primer sets, w/the PGS1 reaction producing a band of ~250bp and the PGS2 reaction producing a band of ~750bp. Colonies 18 and 28 are an exception to this and produced no band with the PGS2 primer set. NTCs were clean. The positive controls worked as expected, yielding a band of ~250bp for PGS1 and a band of ~250bp for PGS2.

It is confusing as to why the size of the PGS2 positive control is different than the product that was generated from the colony PCRs.

Will select 10 colonies for mini-preps.