Tag Archives: DNA

Bisulfite NGS Library Prep – Bisulfite Conversion & Illumina Library Construction of C.gigas larvae DNA

Bisulfite Conversion

The previous attempt at constructing a library for the 1000ppm larvae samples failed. I had previously sheared, quantified, and concentrated the DNA from this sample. As I had done previously, I combined 50ng from each of the two 1000ppm samples for a total of 100ng, and brought the sample volume up to 24μL with NanoPure H2O.

Bisulfite conversion was performed with the Methylamp DNA Modification Kit (Epigentek) according to the manufacturer’s protocol.

Sample was eluted with 10μL of Buffer R6 for immediate use.

 

Library Prep

Bisulfite Illumina library was made with EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina) (Epigentek).  Changes to the manufacturer’s protocol:

  • Samples were transferred to 1.5mL snap cap tubes for all magnetic bead steps in order to fit in our tube magnets.
  • Skipped Step 7.1 (per manufacturer’s recommendation for samples starting with <200ng)
  • Ran 13 cycles during the library amplification step (per manufacturer’s recommendation for samples starting with 100ng)

Sample was transferred to 1.5mL snap cap tube and stored @ -20C.  Will quantify library on Monday when Jake is also finished with his 12 libraries.

 

DNA Bisulfite Conversion – C.gigas larvae OA Sheared DNA

After discussing with Steven, decided to use only samples from 20110513, due to high algae amounts present in the 20110519 samples.

Pooled the DNA of the 400ppm samples (6B2 & 6B5) and pooled the DNA of the 1000ppm samples(1B1 & 1B2) , for a total of two samples. 50ng of each sample was used to make the pools, for a toal of 100ng of DNA in each pool. Calculations are below:
https://docs.google.com/spreadsheets/d/1e7EF05akWeBtO7Xz0UWXhIzRlkVU5HA_rjCE3c4SPEw/edit?usp=sharing

Each pooled sample was brought up to a final volume of 24μL for use in bisulfite conversion kit.

Performed bisulfite conversion of sheared DNA from 20150113 using the Methylamp DNA Modification Kit (Epigentek) according the manufacturer’s protocol.

Samples were eluted with 10μL of Buffer R6 and stored @ -20C.

 

SpeedVac – C.gigas larvae OA DNA

The DNA extracted and sheared on 20150109 was in a volume too great to proceed with bisulfite conversion.  Dried the samples to complete dryness in a SpeedVac (~4hrs).  Reconsitituted DNA in 24μL of NanoPure water.  Will bisulfite convert tomorrow.

Sample list:

SAMPLE ID DATE TREATMENT (ppm) # LARVAE TOTAL DNA (ng)
6B5 20110513 400 5,000 105
1B2 20110513 1000 5,000 109
6B2 20110513 400 10,000 117
1B1 20110513 1000 10,000 593
1B1 20110519 1000 NA 500
1B2 20110519 1000 NA 500
6B2 20110519 400 NA 269
6B1 20110519 400 NA 345

 

Updated the quantification spreadsheet with a column labeled “[New](ug/uL)” that calculates the new concentrations of the above samples in the 24μL volume.

https://docs.google.com/spreadsheets/d/1e7EF05akWeBtO7Xz0UWXhIzRlkVU5HA_rjCE3c4SPEw/edit?usp=sharing

 

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).

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…

qPCR – Taylor Water Filter DNA Extracts from 20120322

Ran qPCR on the Taylor water filter DNA extracts from 20120322 using V.tubiashii VtpA primers (provide by Elene; no SR ID?) instead of 16s primers, which failed to produce acceptable results in the melt curves (see 20120323). Additionally, Elene has a standard curve for V. tubiashii (from 1/12/2011) based off of CFUs/mL, which will allow us to quantify theoretical number of V.tubiashii CFUs present in each sample.

Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Results:

qPCR Date File (CFX96)

qPCR Report (PDF)

Overall, the run looks excellent. Both negative controls and no template controls are clean. Since I was able to use a standard curve, I determined CFUs of V.tubiashii in each sample, as follows:

Mean CFUs per qPCR reaction / template volume per qPCR reaction x filter extraction elution volume (100uL) = total CFUs on water filter.

Total CFUs on filter / filtered water volume = CFUs per mL in Taylor tanks

158 – 16500 copies/2uL = 8250 copies/uL x 100uL = 825000 copies on water filter/1000mL = 825 copies/mL

200 – 5700 copies/2uL = 2850 copies/uL x 100uL = 285000 copies on water filter/1000mL = 285 copies/mL

279 – 325000 copies/2uL = 162500 copies/uL x 100uL = 16250000 copies on water filter/1000mL = 16250 copies/mL

313 – 152 copies/2uL = 76 copies/uL x 100uL = 7600 copies on water filter/1000mL = 7.6 copies/mL

341 – 124000/2uL = 62000 copies/uL x 100uL = 6200000 copies on water filter/1000mL = 6200 copies/mL

410 – 132000/2uL = 66000 copies/uL x 100uL = 6600000 copies on water filter/1000mL = 6600 copies/mL

433 – 63700/2uL = 31850 copies/uL x 100uL = 3185000 copies on water filter/1000mL = 3185 copies/mL

503 – 110/2uL = 55 copies/uL x 100uL = 5500 copies on water filter/1000mL = 5.5 copies/mL

551 – 2000/2uL = 1000 copies/uL x 100uL = 100000 copies on water filter/1000mL = 100 copies/mL

604 – 272/2uL = 136 copies/uL x 100uL = 13600 copies on water filter/1000mL = 13.6 copies/mL

Sample #410 was from the only tank that exhibited mortalities and was the only group of oyster larvae that showed any expression from the V.tubiashii genes (see DATE).

qPCR – Repeat of qPCR from Earlier Today

Repeated exactly what was done earlier today due to apparent contamination in negative controls.

Results:

qPCR Date File (CFX96)

qPCR Report (PDF)

Essentially the same results as the previous run. No template controls do amplify, but EXTREMELY weak and late. Melt curve analysis shows that the signals for the no template controls don’t cross the threshold set by the software.

However, I just looked back at the qPCR results from 20120208 where I used these V. tubiashii 16s primers and realized I got the same results from the cDNA (double-peaks in melt curves and amplification in the no template controls)!! So, I suspect that this primer set isn’t that useful. Will have to examine other sets of V. tubiashii 16s primers to use. Will discuss with Steven.

qPCR – cDNA from 20120208

Performed qPCR on all 12 samples. Used the following primers, provided by Elene, to detect V.tubiashii expression:

  • rseA_F/R
  • VtpA_F/R
  • VtpR_F/R

Used RE22 DNA (provided by Elene) as a positive control. Master mix calcs are the same as yesterday’s qPCR, but using the primers mentioned above. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). All samples were run in duplicate.

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

Positive control worked in all primer sets. All no template controls were clean for all primer sets.

Only one sample (#411) produced any amplification. Amplification was detected in the vtpA primer set (mean Cq = 38.06). However, there was also amplification detected in one of the two replicates for sample #411 in the rseA primer set (Cq = 39.09).