Tag Archives: DNA Isolation

DNA Isolation – C.gigas Larvae from Katie Latterhos

Since the previous isolation attempt was unsuccessful (see 20140922), we’re trying a slightly different approach than yesterday.

Today, I will pellet the samples, remove the RNA Later and then proceed with the Quick-gDNA MicroPrep Kit (ZymoResearch) according to the manufacturer’s protocol for Cell Suspensions and Proteinase K Digested Samples.

Isolated gDNA from two C.gigas larvae samples from Katie Latterhos:

B1 400 D6

B6 D00

Pelleted the samples at 10,000g, 5mins, RT. Although no pellets were visible in either sample, the B1 400 D6 sample did have visible cells/debris at the top of the RNA Later after spinning! So, I recovered that portion of the sample for use in the DNA isolation. The B6 D00 sample had no visible debris, nor pellets, so the RNA Later supernatant was removed and discarded.

Both samples were then processed with the Quick-gDNA MicroPrep Kit (ZymoResearch) according to the manufacturer’s protocol for Cell Suspensions and Proteinase K Digested Samples.

Samples were eluted with 10uL of elution buffer and spec’d on a NanoDrop1000 (ThermoFisher). The 2uL used for each sample were recovered from the NanoDrop.

Results:

Note: The B1 400 D6 sample was spec’d twice, due to an error message on the NanoDrop when spec’ing it the first time. Thus, the second entry for B1 400 D6 is the correct value.

Although the B1 400 D6 sample actually yielded gDNA today, the yield is far too low for use in RAD sequencing (need 500ng; B1 400 D6 yielded only ~260ng). Additionally, the quality of the DNA isolated is horrible (OD 260/280 = 0.81).

The B6 D00 did not yield any DNA.

Will let Steven know and see how he wants to proceed.

DNA Isolation – C.gigas Larvae from Katie Latterhos and Emma

Isolated gDNA from two C.gigas larvae samples (stored in RNA Later) from Katie Latterhos:

B4 400 D05

B6 400 D03

and two samples from Emma:

280E

380E

Emma’s samples were in her -80C box (in rack #2): C.gigas larvae – NOAA O.A. September 2010 Emma

Note: No visible larvae present in either of Katie Latterhos samples. Easily visible larvae in each of Emma’s samples.

DNA was isolated using the Quick-gDNA MicroPrep Kit (ZymoResearch) according to the manufacturer’s protocol for Cell Suspensions and Proteinase K Digested Samples.

Samples were eluted with 10uL of elution buffer and spec’d on a NanoDrop1000 (ThermoFisher). The 2uL used for each sample were recovered from the NanoDrop.

Results:

Not surprisingly, the samples from Katie Latterhos yielded, essentially, no gDNA. Will discuss with Steven.

*UPDATE 20141030*

Steven sent me this screen cap of Emma’s notebook so we could track where the samples originated from

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).

Results:

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.

Results:

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.

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.

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 Isolation – Mackenzie’s C.gigas Gonad Sample

Mac’s been having some difficulty getting good quality gDNA from some of her gonad samples, so she asked me to give it a shot.

Isolated DNA from 10mg (0.010g) of C.gigas gonad tissue using the Qiagen Blood & Tissue DNeasy Kit, with the following changes:

  1. Incubated sample in Buffer ATL + Proteinase K @ 56C for 3hrs

  2. Eluted sample in 100uL of Buffer AE.

Spec’d on NanoDrop1000.

Results:

DNA looks good, both 260/280 ratio and yield. The 260/230 ratio isn’t perfect, but it’s much better than what Mac was seeing. After showing her this, she’s decided to have me isolate DNA from the rest of her samples.

DNA Isolation – C.gigas Female Gonads (from frozen)

Isolated gDNA from Claire’s “Female DNA” (from 05/16/2013) using the Qiagen Blood & Tissue DNeasy Kit according to the manufacturer’s protocol, with the following changes:

  1. Incubated sample in Buffer ATL + Proteinase K @ 56C for 3hrs. Vortexed once each hour.

  2. Eluted with 100uL of Buffer AE.

Results:

Excellent yield and quality is good, although both the 260/280 and 260/230 ratios are on the high side. However, these high values could be an artifact of the high sample concentration (this is a common “issue” with the NanoDrop).

DNA Isolation – Geoduck

Isolated additional geoduck gDNA from the two fresh (now frozen) geoduck’s that Brent provided me with on 20140212 so that we can potentially isolate RNA from the same geoducks to tie in with the DNA Illumina sequencing that Axa will be conducting. Isolated DNA using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol (incubated minced siphon tissue at 56C for 3hrs). Eluted with 75uL of ddH2O and spec’d on NanoDrop1000.

Note: Initial specs were too low for Axa’s requirements (50uL, >= 500ng/uL). SpeedVac’d samples to concentrate, brought volume to 55uL and then spec’d on NanoDrop1000.

Results:

Samples look good. Will send Axa 50uL of all samples, excluding GD01 since that sample is below his desired concentration AND I believe he probably doesn’t want to wait for this DNA any longer.

DNA Isolation – Geoduck

Since yesterday’s DNA isolation failed to yield sufficient quantity of DNA from the ethanol-fixed samples, I isolated additional DNA from the same samples.

Isolated gDNA from 6 geoduck siphon samples provided by Brent using the Qiagen DNeasy Spin Kit. Samples were as follows:

  • Langley 006
  • Langley 007
  • Langley 008
  • Langley 009

The Langley samples were fixed in ethanol in 2006. Geoduck 01/02 were fresh geoduck siphon samples, taken from two live, juvenile geoduck (the rest of the bodies were frozen at -80C).

The samples were processed according to the Qiagen protocol (but utilized an overnight incubation at 56C with Proteinase K), eluted in 50uL of Buffer AE, combined with the corresponding DNA from yesterday, mixed throughly and spec’d on a NanoDrop1000.

Results:

Now have sufficient quantity of DNA for all four of these samples. Will contact Axa (the person who this DNA is intended for) to see if he requires a specific concentration/volume.