Tag Archives: gel

PCR – Sea Pen luciferase

Ran a PCR to obtain luciferase DNA for sequencing.

Used sea pen gDNA extracted by Jonathan on 20150527.


1604 Rr_46_65F
1603 Rr_887_868R

Master mix calcs are here: 20150702_seapen_PCR

Cycling params:

  1. 95C – 10mins
  2. 95C – 15s
  3. 55C – 15s
  4. 72C – 1min
  5. Go to Step 2 39 times


Ran samples on 0.8% agarose, low TAE gel stained with EtBr.





Lane 1 – ladder

Lane 2 – empty

Lane 3 – sea pen gDNA

Lane 4 – NTC


PCR did not work. Was expecting a band of ~800bp.

Looks like I may have overloaded the PCR reaction with gDNA. Used 10μL of gDNA.

However, that is quite the smear, suggesting a significant amount of degradation present in the gDNA.

Will re-run this PCR next week with less gDNA (or, cDNA instead) in order to generate a PCR product.

Bioanalyzer – Geoduck Gonad RNA Quality Assessment

Before proceeding with transcriptomics for this project, we need to assess the integrity of the RNA via Bioanalyzer.

RNA that was previously isolated on 20150508, 20150505, 20150427, and 20150424 (those notebook entries have been updated to report this consolidation and have a link to this notebook entry) were consolidated into single samples (if there had been multiple isolations of the same sample) and spec’d on the Roberts Lab NanoDrop1000:

Google Sheet: 20150528_geoduck_histo_RNA_ODs

NOTE: Screwed up consolidation of Geoduck Block 03 sample (added one of the 04 dupes to the tube, so discarded 03).

RNA was stored in Shellfish RNA Box #5.

RNA was submitted to to Jesse Tsai at University of Washington Department of Environmental and Occupational Health Science Functional Genomics Laboratory for running on the Agilent Bioanalyzer 2100, using either the RNA Pico or RNA Nano chips, depending on RNA concentration (Pico for lower concentrations and Nano for higher concentrations – left decision up to Jesse).



Bioanalzyer 2100 Pico Data File (XAD): SamWhite_Eukaryote Total RNA Pico_2015-05-28_12-50-00.xad
Bioanalzyer 2100 Nano Data File (XAD): SamWhite_Eukaryote Total RNA Nano_2015-05-28_13-22-53.xad


Pico Gel Representation


Pico Electropherogram


Nano Gel Representation


Nano Electropherogram


Jesse alerted me to the fact that they did not have any ladder to use on the Nano chip, as someone had used the remainder, but failed to order more. I OK’d him to go ahead with the Nano chip despite lacking ladder, as we primarily needed to assess RNA integrity.


Bad Samples:

  • Geo 04 – No RNA detected
  • Geo 65, 67, 68 – These three samples show complete degradation of the RNA (i.e. no ribosomal band present, significant smearing on the gel representation).

All other samples look solid. Will discuss with Steven and Brent on how they want to proceed.

Full list of samples for this project (including the Block 03 sample not included in this analysis; see above). Grace’s notebook will have details on what the numbering indicates (e.g. developmental stage).

  • block 02
  • block 03 (no RNA)
  • block 04 (no RNA)
  • block 07
  • block 08
  • block 09
  • block 34
  • block 35
  • block 38
  • block 41
  • block 42
  • block 46
  • block 51
  • block 65 (degraded RNA)
  • block 67 (degraded RNA)
  • block 68 (degraded RNA)
  • block 69
  • block 70

Bioanalyzer Data – Geoduck RNA from Histology Blocks

I received the Bioanalyzer data back for the geoduck foot RNA samples I submitted 20150422. The two samples were run on the RNA Pico chip assay.



Bioanalzyer 2100 Data File (XAD): SamWhite_Eukaryote Total RNA Pico_2015-04-23_13-04-16.xad

Data file requires 2100_Expert_B0208_SI648_SR2 version of the software (Windows).

Gel Representation




The samples look really good! As we’ve seen previously in shellfish RNA, there is a single, prominent rRNA band/peak with very little degradation (smearing and/or no prominent peak/band).

Will proceed with RNA isolation from the remaining histology blocks.

Gel – Sheared LSU C.virginica Oil Spill gDNA (from yesterday)

Ran ~250ng of sheared C.virginica gDNA from yesterday’s shearing.


Ladder used: O’GeneRuler 100bp Ladder (ThermoFisher)

The shearing is, surprisingly, very inconsistent across the samples. The target average fragment size was ~350bp. However, most of these samples are <250bp. The MethylMiner Kit (Invitrogen) suggests that an average fragment length of 100 – 200bp is ideal for short-read high-throughput sequencing, but we’re going to perform a bisulfite conversion on these which will result in some additional fragmentation, further reducing the average fragment size. Will proceed with methylated DNA enrichment.

Gel – Sheared gDNA

Ran ~250ng (out of 3000ng, according to Claire) of LSU C.virginica oil spill gDNA on a gel that was previously sheared by Claire to verify that shearing was successful.

Ran unsheared side-by-side with sheared gDNA for comparison.

Note: HB16 and NB3 did not have any unsheared gDNA left in their tubes, so nothing was run on a gel.


Ladder used: O’GeneRuler 100bp Ladder (ThermoFisher)

Well, it’s rather obvious that the initial shearing did NOT work. Will re-shear the samples.

UPDATE: Looking at the Biorupter (Diagenode) manual, it turns out that shearing samples in a 1.5mL tube (in which these were sheared) requires a minimum volume of 100uL. All the samples were far below this minimum volume. Additionally, the recommendations in the manual to reach the target size range are significantly longer (30 – 40 cycles) than what was applied (4 cycles). The combination of these two factors are likely the reason that shearing didn’t take place.

DNA Shearing & Size Selection – Oly Oyster gDNA RAD P1 Adapters (from 20141105)

Pooled “low quality” samples and pooled “high quality” samples separately (in 1.5mL snap cap tubes) prior to shearing to improve chances of getting similar DNA size ranges.

Samples were selected based on the gels run by Steven on Oct. 17, 2014: http://sr320.tumblr.com/

Low quality samples (5uL from each):

All rows, columns 1 -9

Higher quality samples (5uL from each):

All rows, columns 10 -12

Sheared each samples with the following cycling protocols on the Biorupter Plus (Diagenode):


  • 3 cycles of:
  • 30 seconds on
  • 59 seconds off



4 cycles of:

  • 30 seconds on
  • 59 seconds off


Ran a subset of sheared gDNA (5uL from each pool) on gel to verify final size range:

Gel loading:

Lane 1 – O’GeneRuler 100bp Ladder (ThermoFisher)

Lane 2 – Low quality

Lane 3 – Higher quality

I neglected to run a set of un-sheared DNA.

Both samples appear to have an average size of 200 – 400bp.

After confirming satisfactory shearing, the two samples were combined and run on a 1% agarose low TAE gel (stained with EtBr) for size selection.

O’GeneRuler 100bp Ladder (ThermoFisher)

O’GeneRuler 100bp Ladder (ThermoFisher)

Size range of sheared DNA from 300 – 500bp was excised from gel.


Gel fragment weighed 254mg.

Purified using MiniElute Gel Extraction Kit (Qiagen).

Added three volumes (762uL) of Buffer QG to gel slice.

Incubated ~10mins on rotator until gel slice was fully dissolved.

Added one gel slice volume (254uL) of isopropanol; inverted multiple times to mix.

Added 700uL to MiniElute column; spun max speed (~16,000g) 1min; discarded flow-through.

Added remainder of sample to MiniElute column; spun max speed (~16,000g) 1min; discarded flow-through.

Added 500uL of Buffer QG to MiniElute column; spun max speed (~16,000g) 1min; discarded flow-through.

Added 750uL of Buffer PE to MiniElute column; incubated @ RT for 5mins; spun max speed (~16,000g) 1min; discarded flow-through.

Spun MinElute column spun max speed (~16,000g) 1min; transferred column to clean 2.0mL tube.

Added 50uL of Buffer EB to column, incubated @ RT for 5mins and spun max speed (~16,000g) 1min; discarded column.

Stored sample @ 4C.

PCR – Mac’s Bisulfite-Treated DNA

Realized that the PCR performed on 20140828 used the incorrect forward primer! As such, am repeating as before, but with the correct forward primer:

CgBS_733_26796F (SRID: 1597)

NOTE: Nothing left of sample EV2.28 bisulfite, so this was not run.


Ladder – O’GeneRuler 100bp Ladder (ThermoFisher)

Samples are loaded in numerical order from left to right, with a NTC sample before the second ladder.

All samples ran at ~275bp, which is larger than the previous gels. Confirmed with Mac that this gel looks correct. Will contact Cassie at Fred Hutchinson to go forward with PyroMark sequencing of these products.

Evidently, it would seem that Mac (and I) used the incorrect primer set when performing this PCR most recently. Doh!

PCR – Mac’s Bisulfite-Treated DNA

Per Mac’s request, ran a PCR on a set of bisulfite-treated DNA (in her gDNA 2014 box in small -20C):

  • EV2.16 bisulfite
  • EV2.20 bisulfite
  • EV2.22 bisulfite
  • EV2.24 bisulfite
  • EV2.28 bisulfite
  • EV2.29 bisulfite
  • EV2.32 bisulfite
  • EV2.33 bisulfite

DNA needed to be diluted. Diluted according to this sheet provided by Mac:


NOTE: EV2.28 didn’t have sufficient DNA left to prepare the dilution according to Mac’s sheet. Instead, the remaining volume ofEv2.28 bisulfite DNA (0.5uL) was diluted in a total volume of 2.5uL to maintain the same dilution ratio.

Master mix calcs are here: 20140828 – PCR Mac Bisulfite Samples

Primers used were:

CgBS_733_26796Seq (SRID: 1598)
CgBS_733_26796R_5’biotin (SRID: 1596)

Cycling params:

  1. 1. 95C – 10mins
  2. 2. 94C – 30s
  3. 3. 56C – 30s
  4. 4. 72C – 30s
  5. 5. Repeat steps 2 – 5 44 more times
  6. 6. 72C – 10mins


Ladder used is O’GeneRuler 100bp DNA Ladder (ThermoFisher).

According to Mac, the expected band size is ~300bp. However, all samples are running at ~150bp. Mac is confused and does not know what to do.

*UPDATE 20140902* – Realized I used the wrong forward primer! Will repeat PCR with correct primer. Wonder if Mac did the same thing…

DNA Isolation – Mackenzie’s C.gigas EE2 Gonad Samples

Isolated DNA from the following samples, provided by Mackenzie:

  • EE2v2, 22.go
  • EE2v2, 20.go
  • EE2v2, 28.go
  • EE2v2, 29.go
  • EE2v2, 16.go
  • EE2v2, 32.go
  • EE2v2, 24.go
  • EE2v2, 33.go

Samples were suspended in 500uL of DNazol (Molecular Research Center), 5uL of PolyAcryl Carrier (Molecular Research Center), 2.75uL Proteinase K (Fermentas; 18.5mg/mL stock), briefly vortexed and incubated 24hrs at RT on rotator. Samples were briefly vortexed and insoluble material was pelleted 10,000g, 10mins, RT. Supe was transferred to fresh tube, mixed with 250uL of 100% EtOH, incubated at RT 5mins, and DNA was pelleted by spinning samples 5,000g, 5mins, RT. Supe was discarded, pellets washed with 1mL of 70% DNazol/30% EtOH solution. Supe was discarded and pellets were washed with 1mL 70% EtOH. Pellets were stored @ -20C under 95% EtOH over the weekend. Supe was discarded and pellets were washed with 70% EtOH. This step was repeated 2 more times. Supe was discarded and pellets were resuspended in Low TE Buffer, spec’d on NanoDrop1000 and run on a gel (10uL of each sample).


Yields look good and OD260/280 values look excellent. Most of the OD260/230 values aren’t good, but they rarely are.

Gel Loading:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – EV2 16.go

Lane 3 – EV2 20.go

Lane 4 – EV2 22.go

Lane 5 – EV2 24.go

Lane 6 – EV2 28.go

Lane 7 – EV2 29.go

Lane 8 – EV2 32.go

Lane 9 – EV2 33.go

Lane10- Hyperladder I (Bioline)

All samples (excluding EV2 22.go) look pretty good, with minimal smearing. All samples exhibit low molecular weight smear which is either degraded DNA or residual RNA carryover. EV2 22.go had very little tissue, so yields were expected to be extremely low. However, I was anticipating to be able to visualize it on the gel (loaded 10uL = ~90ug).

DNA Isolation – Claire’s C.gigas Female Gonad

Trying this sample again(!!), but will now use TE for pellet resuspension to prevent sample degradation. Incubated sample RT on rotator in 500uL of DNazol + 2.7uL of Proteinase K (Fermentas; Stock 18.5mg/mL) for 5hrs. Added additional 500uL of DNazol, mixed gently and followed DNazol manufacturer’s protocol. Performed first pellet was with 70% DNazol/ 30% EtOH solution. Resuspended pellet in 200uL of TE and spec’d on NanoDrop1000.


Yield is good. 260/280 value is good. 260/230 value is poor. Will run on gel to evaluate integrity.

Loaded 10uL (~830ng) on 1.0% agarose 1x modified TAE gel stained with EtBr.

Gel Loading Guide:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – C.gigas female gonad gDNA (CgF)

Well, this certainly looks much better than previous preparations, in that there is an obvious high molecular weight band present (previously, this had been absent). The low molecular weight bands/smears are possibly RNA carryover and/or degraded DNA. Will discuss with Steven and then, most likely, bring downtown for Illumina sequencing.

UPDATE 20140508: Downtown sequencing facility says there’s only ~800ng of DNA! This is a far cry from the minimum amount needed for sequencing (6ug). Looking at the gel above and comparing sample band intensity to the ladder band intensities suggests that the downtown sequencing facility is correct. I loaded 10uL of DNA on the gel and the intensity of the high molecular weight band is similar to the 400bp band intensity. This corresponds to 40ng of DNA. That means the CgF gDNA band is 40ng/10uL = 4ng/uL. I resuspended the gDNA pellet in 200uL of TE, so 200uL x 4ng/uL = 800ng; exactly what the sequencing facility says they measured…

I’m not entirely sure what is happening here. Until very recently, there were almost never such egregious differences between the NanoDrop measurements and what they were measuring downtown at the sequencing facility. It seems as though they have changed the way they quantify samples (possibly using an Agilent Bioanalyzer instead of the Life Technologies Qubit fluorometer?), but this doesn’t mean their measurements are incorrect. However, I’m starting to suspect that the reason the initial sequencing of this sample was due to an overestimation of the quantity of input DNA (since I believe they were still using the fluorometer back then).

As such, it’s become clear that C.gigas gonad samples seem to yield poor quantities of gDNA, relative to the amount of input material. Additionally, there may be insufficient sample left to generate a useable quantity of gDNA to complete this resequencing effort.