20100630

MeDIP - SB/WB Fragmented gDNA (from 20100625)

After confirming proper fragmentation (~460bp average fragment size) via Bioanalyzer earlier today, began the MeDIP process. Brought fragmented DNA samples up to 350uL with TE. Heated samples @ 95C for 10mins, then incubated on ice 5mins. Added 100uL of 5x MeDIP Buffer (50mM Na2HPO4, 700mM NaCl, 0.25% Triton-X 100), 45uL of TE and 5uL (5ug) of anti-methyl cytidine antibody (Diagenode; 5-mC monoclonal antibody cl. b). Incubated O/N, 4C rotating.



Bioanalyzer - Fragmented SB/WB gDNA (from 20100625)

To gain a more quantitative assessment of the fragmentation from 20100625, I ran 1uL of each sample (~55ng, according to pre-fragmentation spec values) on the Agilent Bioanalyzer 2100, using the DNA 1000 kit, according to manufacturer's protocol.

Results:
Avg. size of fragmentation is ~460bp for the two samples. Fragmentation size was determined by marking the same region on both sample's electropherograms (see below).


R37: Avg. size = 450bp (in Region 1, marked with blue lines in image below)

external image 20100630%20Bioanalyzer%20R37%20Frag%20electropherogram.jpg




R51: Avg. size = 468bp (in Region 1, marked with blue lines in image below)

external image 20100630%20Bioanalyzer%20R51%20Frag%20electropherogram.jpg




Overlay of R37 and R51 fragmentation. Note that both electropherograms are nearly identical (this is good).

external image 20100630%20Bioanalyzer%20R37_R51Frag%20electropherogram%20overlay.jpg







20100625

gDNA Sonication - SB/WB gDNA pools (prep for MeDIP) from 20100618

The previous attempt at sonication (see 20100618) failed, likely due to no using the correct equipment (tubes and Covaris adapter). The two gDNA pools, which had previously been unsuccessfully fragmented on 20100618 (SB and WB) were sonicated using a Covaris S2. Used the guidelines of the manufacturer (listed below) for shearing gDNA to a desired target size (500bp):

Duty Cycle: 5%
Intensity: 3
Cycels per Burst: 200
Time (seconds): 90
Temp (water bath): 4C
Power Mode: Frequency Sweeping
Sample Volume: 120uL
Buffer: TE
DNA Mass: ~8ug
Starting Material: >50kb
AFA Intensifier tubes and associated Covaris adapter.


After shearing, ran 250ng of each pool on a 2% TAE agarose gel for fragmentation verification.


Results:

external image 20100625.jpg


Lane 1 - Hyperladder I
Lane 2 - R37
Lane 3 - R51

Looking at this gel, the samples have been successfully fragmented and I would estimate have generated and average fragment size of ~400bp (going from bottom to top of the Hyperladder: 200bp, 400bp, 600bp, 800bp, 1000bp). So, this looks great! Can proceed with remainder of MeDIP procedure at any time.

Additionally, I will confirm a more accurate assessment of average fragment size by running these two samples on the Agilent Bioanalyzer.


=

20100624 =

Samples Received - Hard Clam samples from Rutgers and MBL *Important Note: These were received while I was out of lab. This notebook entry was added 20101021*

Received sets of gill tissue and hemolymph in RNA Later from Rutgers (Emily). Here's the note that was included with the samples.

Received set of gill tissue in RNA Later MBL (Scott Lindell).

All samples were stored @ -80C.


20100618

gDNA Sonication - SB/WB gDNA pools (prep for MeDIP) from earlier today

The two gDNA pools (SB and WB) were sonicated using a Covaris S2. Used the guidelines of the manufacturer (listed below) for shearing gDNA to a desired target size (500bp):

Duty Cycle: 5%
Intensity: 3
Cycels per Burst: 200
Time (seconds): 90
Temp (water bath): 4C
Power Mode: Frequency Sweeping
Sample Volume: 120uL
Buffer: TE
DNA Mass: ~8ug
Starting Material: >50kb

To be noted, the Covaris guidelines list the use of an "AFA Intensifier" tube, which I did not use (because we don't have them).

After shearing, ran 250ng of each pool on a 2% TAE agarose gel for fragmentation verification. Also ran 250ng of pre-sonication DNA from each pool as controls.

Results:

external image 20100618%20sonicated%20DNA%20gel.jpg

Lane 1 - Hyperladder I
Lane 2 - R37, Un-sonicated
Lane 3 - R37, sonicated
Lane 4 - R51, Un-sonicated
Lane 5 - R51, sonicated


Sonication with the Covaris did NOT produce the desired fragmentation (500bp smear) in either sample, although the R37 sonicated samples shows a significantly greater degree of fragmentation than the R51 sonicated sample. Not sure how to explain this difference, other than the R51 sample has a greater amount of DNA. Additionally, the results could be explained by the fact that we did not use the AFA Intensifier listed in the Covaris guidelines...

Am consulting with a person in Genome Sciences who has used a Covaris for DNA fragmentation in the past to see if the AFA Intensifiers are indeed necessary and, if so, we can use two of them. Hopefully have an answer soon and be able to proceed with additional fragmentation next week.


gDNA Precipitation - SB/WB gDNA pools (prep for MeDIP)

8 gDNA samples from SB were pooled and 8 gDNA samples from WB were pooled, using equal amounts of gDNA from each sample (1250ng) for a total of 10ug (see SB/WB Mac's MeDIP spreadsheet for specific samples/volumes used in pooling). Since samples were stored in pH-adjusted NaOH (see 20100605), they needed to be precipitated in order to have the gDNA suspended in TE for the downstream steps of methylated DNA immunoprecipitation (MeDIP). 10% 3M sodium acetate (pH = 5.2) was added to each tube, then 2.5 vols of 100% EtOH and mixed. Samples were incubated @ -20C for 30mins. DNA was pelleted by spinning 16,000g for 30mins @ 4C. Supe was discarded. Pellets were washed with 1mL 70% EtOH and then pelleted @ 16,000g for 10mins @ 4C. Supe was discarded and gDNA was resuspended in 120uL of TE (pH = 8.0) and spec'd.

Results:

external image 20100618%20pooled%20gDNA.JPG

The R37 (SB) sample pool yielded 7.056ug after precipitation and the R51 (WB) sample pool yielded 8.834ug after precipitation (started with 10ug). This is good, as 6ug is needed for MeDIP and I wanted to have some (~250ng) available for running as an un-sonicated control on the post-sonication gel. Will transfer 250ng from each pool to separate tubes and then proceed with sonication.



20100608

gDNA Isolation - Mac gigas larvae samples: control larvae 6.7.10 and 5-aza tr larvae 6.7.10

Continued gDNA isolation of the above mentioned larvae samples that was started by Mac yesterday. Amount of larvae in tubes looked disproportionately large, relative to the amount of DNAzol used in the O/N Proteinase K digestion(~500uL) so I added and additional 500uL of DNAzol to each of the two samples and gently pipetted a few times to mix.

Pelleted residual tissue 10mins @ 10,000g @ RT. Transferred supe to new tubes. Precipitated DNA with 0.25mL 100% EtOH. Incubated 3mins @ RT. DNA was pelleted 5mins @ 5000g @ RT. Supe was removed, pellets were washed with 1mL 75% EtOH (x2). Supe was fully removed and the DNAs were resuspended in 800uL 8mM NaOH (made by Amanda Davis 5/20/10).

1M HEPES (provided with DNAzol) was added at a 1:100 dilution to achieve a pH = 8.0. This was based on the DNAzol protocol calculations (For 1mL of 8mM NaOH, use 101uL of 0.1M HEPES = pH 8.0).

Samples were spec'd on NanoDrop 1000 on 20100607. Used a sample with 8mM NaOH and 1M HEPES to match the pH = 8.0 of the samples.

Results:

external image 20100608%20gDNA.JPG

Yields are very good and the 260/280 ratios are pretty good. The 260/230 ratios are very poor and is likely due to the large amount of larvae used in the procedure. Will run samples on a gel to evaluate DNA integrity. *UDPATE: Mac ran these samples on 6/9/10 (see her notebook on that date) and they look perfect.*



20100605

gDNA Isolation - Mac gigas gill samples (continued from yesterday)

Continued with gDNA isolation from yesterday's samples. Samples were gently pipetted up and down to further dissolve remaining tissue, although tissue did not dissolve entirely. Pelleted residual tissue 10mins @ 10,000g @ RT. Transferred supe to new tubes. Precipitated DNA with 0.25mL 100% EtOH. Incubated 3mins @ RT. DNA was pelleted 5mins @ 5000g @ RT. Supe was removed, pellets were washed with 1mL 75% EtOH (x2). Supe was fully removed and the pellets were resuspended in 200uL 8mM NaOH (made by Amanda Davis 5/20/10).

1M HEPES (provided with DNAzol) was added at a 1:100 dilution to achieve a pH = 8.0. This was based on the DNAzol protocol calculations (For 1mL of 8mM NaOH, use 101uL of 0.1M HEPES = pH 8.0).

Samples were spec'd on NanoDrop 1000 on 20100607. Used a sample with 8mM NaOH and 1M HEPES to match the pH = 8.0 of the samples.


Results:


external image 20100607%20gDNA-01.JPG

Overall DNA quality looks good (based on 260/280 ratios). Yields seem satisfactory. Will run samples on gel to verify gDNA integrity (see below).



250ng of each sample was run on a 1.2% TAE agarose gel. Gel was run on 20100607.

external image 20100607.jpg
The results are pretty interesting.

Most of the R51 samples are pretty good looking (i.e. high molecular weight band, little smearing), but there are some samples that show a high degree of degradation (e.g. #18, #19).

All of the R37 samples look STELLAR (i.e. high molecular weight band, no smearing)!

The stark differences between the R51 samples and the R37 are intriguing. Although not currently verified (as of 20100607), I suspect that the amount of tissue stored in RNA Later possibly contributes to the long term integrity of the DNA, as nearly all of the R37 samples had very little tissue in the RNA Later. Whereas the R51 tissue samples were significantly larger in virtually every sample. I will do a visual inspection of the tubes to see if there is indeed a correlation between tissue size and apparent DNA quality.




20100604

gDNA Isolation - Mac gigas gill samples


Set up gDNA isolation from the following samples:



Samples were thawed from -80C. Tissue was removed from RNA Later (RNA Later gDNA isolation protocol; this protocol doesn't indicate that anything needs to be done to the sample prior to gDNA isolation) and ~25mg was cut from each and placed in 0.5mL of DNAzol. 2.7uL of Proteinase K (Fermentas; 18.5mg/mL) was added to each tube to reach a final concentration of 100ug/mL. The digests were incubated @ RT O/N.



20100528

gDNA Isolation - Mac gigas gill samples (continued from yesterday)

Continued with gDNA isolation from yesterday's samples. Additionally, isolated gDNA from R51 01, but homogenized the tissue (using disposable 1.5mL mortar/pestle) in 0.5mL of DNAzol and topped off to 1.0mL. All 3 samples were gently pipetted up and down to further dissolve the tissue. For those samples that were subjected to Proteinase K digestion, I transferred 100uL of the solution to a new tube containing 1mL of DNAzol, as described in the DNAzol protocol (see "Notes, #5" part of protocol). Tubes were incubated 10mins @ RT.

Pelleted residual tissue 10mins @ 10,000g @ 4C. Transferred supe to new tubes. Precipitated DNA with 0.5mL 100% EtOH. Incubated 3mins @ RT. DNA was pelleted 5mins @ 5000g @ 4C. Supe was removed, pellets were washed with 1mL 75% EtOH (x2). Supe was fully removed and the pellets were resuspended in 8mM NaOH (made by Amanda Davis 5/20/10). See below for volumes:

R51 11 - 50uL
R51 01 - 100uL
R51 01 homogenized - 200uL

1M HEPES (provided with DNAzol) was added at a 1:100 dilution to achieve a pH = 8.0. This was based on the DNAzol protocol calculations (For 1mL of 8mM NaOH, use 101uL of 0.1M HEPES).

Samples were spec'd on NanoDrop 1000. Used a sample with 8mM NaOH and 1M HEPES to match the pH = 8.0 of the samples.

Results:

external image 20100528%20gDNA-01.JPG


The 260/280 ratios for all samples are great. Yields are significantly lower than I was expecting. However, for R51 11 and R51 01, only 100uL (1/5th) of the digestion was used as described in the protocol. I may recover the remainder of the R51 11 gDNA since remaining tissue could be a limiting factor. The homogenized sample had the highest yield (9.35ug), suggesting this may be a more efficient approach to obtaining gDNA (faster procedure and higher yields).

These results also demonstrate that gDNA can be successfully isolated from samples stored in RNA Later (gel pending).

Due to low yields of the R51 11 and R51 01 samples, these will not be run on a gel until the rest of the DNA is isolated.

Isolated the remainder of the gDNA from R51 11 and R51 01. Added an additional 0.5mL of DNAzol to each tube and pipetted up and down to further dissolve the remaining tissue. Then, proceeded as described above. Samples were resuspended in 200uL of 8mM NaOH with 2.02uL of 1M HEPES added.

Results:

external image 20100528%20gDNA-02.JPG

Both samples have great yields and excellent 260/280 ratios. Digestion, combined with gentle pipetting to disrupt undigested tissue, appears to lead to higher yields. However, the process is more time consuming than just basic, physical homogenization of tissue.


external image 20100601-01.jpg

Gel Loading (0.5ug of each DNA was loaded in a volume of 25uL)

Lane 1. Hyperladder
Lane 2. R51 11
Lane 3. R51 01
Lane 4. R51 01 homogenized

The R51 11 sample looks perfect. The other two samples however, appear degraded, with the homogenized sample looking the worst. Interestingly, those are both from the same source tissue, possibly suggesting that the DNA in this particular gill sample is actually degraded and is independent of the preparation since the R51 11 (Lane 2) and R51 01 (Lane 3) were prepared simultaneously and in the same way. The gel also seems to show that physically homogenizing the sample leads to greater degradation than performing the Proteinase K digestion.



20100527

Package Rec'd - From NOAA in Connecticut

Rec'd 6 15mL conical tubes with liquid cultures of various algae. It appears that we rec'd two of each culture. No note/info included with package. Tubes will be stored @ RT in the styrofoam conTube labels are listed below:

Tetraselmis cheri Ply429
Tetraselmis cheri Ply429
Thalassiaosira weissflugii TW
Thalassiaosira weissflugii TW
Isochrysis sp. T-150
Isochrysis sp. T-150


gDNA Isolation - Mac gigas gill samples

Set up gDNA isolation from the following samples:

R51 01 (WB R051-0410-01)
R51 11 (WB R051-0410-11)

Samples were thawed from -80C. Tissue was removed from RNA Later (RNA Later gDNA isolation protocol; this protocol doesn't indicate that anything needs to be done to the sample prior to gDNA isolation) and ~25mg was cut from each and placed in 0.5mL of DNAzol. 2.7uL of Proteinase K (Fermentas; 18.5mg/mL) was added to each tube to reach a final concentration of 100ug/mL. The digests were incubated O/N @ RT with rotation.



20100526

SOLiD ePCR/Templated Bead Prep - Lake Trout Lean library

ePCR was performed following ABI's "full scale" protocol, using 1pM of SOLiD cDNA library.

Templated bead preparation was performed according to the "full scale" protocol.

Bead counts are calculated as follows:

Avg bead count x # hemacytometer squares x volume in hemacytometer (uL) x dilution factor = beads/uL x suspension volume (uL) = total beads

Initial Bead count: (1:200 dilution)

Lean: 126, 138, 122, 138 Avg. = 131

Lean: 131 x 25 x 10 x 200 = 6.55x10^6 beads/uL x 200uL = 1.31x10^9 beads

Templated Bead counts (1:10 dilution)

Lean: 165, 171, 186, 160 Avg. = 170.5

170.5 x 25 x 10 x 10 = 426250 beads/uL x 400uL = 1.705x10^8 beads

Percent Recovery Templated Beads

Lean: (1.705x10^8 beads)/(1.31x10^9 beads) x 100 = 13.02% recovery

Results: Yield is significantly higher than the previous preparation performed with this sample. The percent recovery falls into the desired range of 5-15%, so things look good there, too. Will contact Rhonda and get info regarding when this and the other 7 samples can go on a run.



20100513

SOLiD Templated Bead Prep - Yellow perch CT, WB and lake trout Lean libraries (continued from yesterday)

Templated bead preparation was performed according to the "full scale" protocol.

Bead counts are calculated as follows:

Avg bead count x # hemacytometer squares x volume in hemacytometer (uL) x dilution factor = beads/uL x suspension volume (uL) = total beads

Initial Bead counts: (1:200 dilution)

CT: 132, 133, 127, 136 Avg. = 132
WB: 127, 128, 119, 126 Avg. = 125
Lean: 121, 114, 132, 109 Avg. = 119

CT: 132 x 25 x 10 x 200 = 6.6x10^6 beads/uL x 200uL = 1.32x10^9 beads
WB: 125 x 25 x 10 x 200 = 6.25x10^6 beads/uL x 200uL = 1.25x10^9 beads
Lean: 119 x 25 x 10 x 200 = 5.95x10^6 beads/uL x 200uL = 1.19x10^9 beads


Templated Bead counts (1:10 dilution)

CT: 91, 80, 100, 78 Avg. = 87.25
WB: 69, 70, 75, 65 Avg. = 69.75
Lean: 40, 52, 48, 46 Avg. = 46.5

CT: 87.25 x 25 x 10 x 10 = 218125 beads/uL x 400uL = 8.7525x10^7 beads
WB: 39.75 x 25 x 10 x 10 = 174375 beads/uL x 400uL = 6.975x10^7 beads
Lean: 46.5 x 25 x 10 x 10 = 116250 beads/uL x 400uL = 4.65x10^7 beads


Percent Recovery Templated Beads

CT: (8.7252x10^7 beads)/(1.32x10^9 beads) x 100 = 6.61%
WB: (6.975x10^7 beads)/(1.25x10^9 beads) x 100 = 5.58%
Lean: (4.65x10^7 beads)/(1.19x10^9 beads) x 100 = 3.91%


Results: Everything looks pretty darn good. One mild concern, however, is the yield from the the Lean library. An 8-well slide requires 41 million beads for a run. Additionally, I believe 15 million are needed for a WFA (quality check, pre-run). This means that the Lean prep is nearly 10 million beads short of what is necessary for a "complete" run of this sample. Will send the numbers to Rhonda and see what her opinion is and what she suggests to do. But, based on the percent recovery, all the samples should be really high quality (extremely few polyclonal beads).





20100512

SOLiD ePCRs - Yellow perch CT, WB and lake trout Lean libraries

Performed ePCRs on these samples from DATE, following the "full scale" protocol. A work flow analysis (WFA) run on these samples from the initial ePCRs/templated bead prep (DATE) revealed too many polyclonal beads, thus requiring them to be redone. ePCRs will be performed using 1.0pM (120pg/uL) of the SOLiD cDNA fragment libraries, instead of the 1.5pM (180pg/uL) used previously.

CT -

WB -

Lean -


20100505

Package - Hard Clam gill tissue/hemolymph in RNA later

Rec'd package from Rutgers (Emily Pearson) containing two large ziplock bags on "wet" ice, each of those containing smaller bags with sample tubes in them. One large bag contains gill tissue samples and the other large bag contains hemolyph samples. Samples will temporarily be stored @ 4C until they can be cataloged and boxed by Lexie later today.



DNA Isolation - Qiagen Kit Comparison

Note: This information was added 20140407. Yes, you read that correctly.

Someone had noticed that gDNA isolated using a Qiagen DNeasy Blood & Tissue Kit we rec'd in April 2010 seemed to be yielding degraded DNA.

The two samples used for the comparison were a single tail (split in two equal weight pieces) from a juvenile salmon that was snap frozen, without preservatives, at the time of its collection. The samples were prepped according to the supplied protocol, with each sample being processed with only reagents/columns from the "bad" kit or the "good" kit. 0.5ug of eluted DNA was then run on a 1.2% agarose-TAE gel containing 0.1ug/mL of ethidium bromide. 5uL of Bioline's Hyperladder I was loaded for size assessment (see link for marker layout).

Results:

external image 20100505%20Qiagen%20Kit%20compare%20swhite.jpg

Clearly, there's significant quality difference! A free replacement kit was sent by Qiagen.


20100503

qPCR - V.tubiashii primers test (Vpt A and Vt IGS)

Utilized to sets of primers obtained from the Friedman Lab: VptA (referred to as "Hasegawa", even though the reference article calls the primers Vtp A) and Vt IGS (referred to as "Lee" primers, presumably from a published article). For template, used "RE22 DNA" that was given to me by Elene. Tube is dated 9/10/09 and has no indication of concentration. Performed qPCR on a set of 10-fold dilutions. Plate layout/qPCR set up is here, along with dilution series used.


Results:

The VptA primer set generated a nice looking set of dilutions with appropriate spacing (~3.2 Ct/10-fold dilution). HOWEVER, the raw fluorescence signal is very low (only 0.4 units; good signal is usually 3-5-fold higher) AND the melting curve doesn't look that great. The melting curve could look poor due to the low signal, since it doesn't come up much higher than background levels.

It should be noted that the low fluorescence levels generated could simply be due to the amplicon size generated by these primers. The amplicon size is only 63bp. An amplicon of this size might not be able to incorporate significant amounts of dye to generate a "normal" level of fluorescence (1.25 - 2 units).



The Vt IGS primers failed to generate any product.