20100917
Opticon Calibration
Distributed 50uL of FAM calibration dye to wells. Ran out of dye!!
Looking back at old purchasing logs, it turns out we need 2 orders of dye packs to have enough for a 96-well plate.
Will cap existing plate with dye, wrap in foil and store @ -20C.
Ordered an additional pack of dye (Cat# 10006046; not available online, must call BioRad to order). Will ship on Monday. Will finish calibration procedure on Tuesday (20100928). Ugh.
Results:
Empty Plate:
Plate with dye (presumably calibrated):
After running the calibration protocol with the dye, all the wells should show consistent fluorescence levels. Clearly, they do not. Oddly enough, there appears to be a cyclical pattern across the wells of low -> high -> low fluorescence. The calibration protocol advises that if the wells do not exhibit consistent fluorescence across wells, then the plate should be read again. The graph above is the 2nd reading, which appears to be the same as the 1st. Conclusion is that the Opticon 2 is not working correctly and will contact BioRad for price quotes on repairs.
20100909
qPCR - Hard Clam Primers on cDNA from yesterday
Performed qPCR on Friedman Lab machine targeting immune-related genes in hard clam.
Rough plate layout/master mix calcs are here.
qPCR report from Friedman Lab machine is here (PDF) and shows cycling params, plate layout and Cts.
Results:
CFX96 Data file is here.
The following primer sets failed to produce an amplicon:
Mm_TRAF6
Mercenaria_Rel
TLR
STI
CytP450-like
Raw fluorescence data was extracted (No baseline subtraction) and processed with PCR Miner.
Data workup/analysis is here. Here is a graph of those primer sets producing an amplicon. All were normalized to actin, which exhibited the smallest amount of deviation across all three samples of the normalizing/housekeeping genes analyzed.
As a preliminary run with these genes, there are a number of promising candidates that could yield some interesting data regarding the physiological response of hard clam to exposure to QPX.
20100908
Reverse Transcription - DNased Hard Clam RNA from earlier today
Prepared cDNA using 1ug of RNA from each of the 3 pools (CA, MA, MAX) and processed according to Promega's M-MLV protocol, using oligo dT primers.
Calcs and master mix set up are here. Briefly, RNA was combined with oligo dT primers, denatured @ 70C for 5mins, immediately placed on ice for 2mins, mixed with RT master mix, incubated 1hr @ 42C, 3mins @ 95C, and then stored @ -20C.
DNase - DNasing Hard Clam RNA from yesterday
Pooled 2ug of each sample in each group (MAX, CA, MA) for a total of 6ug of RNA (3 total samples), brought volume up to 50uL and DNased using Ambion's Turbo DNA-free following the rigorous protocol. Calcs can be seen here. Spec'd:
Results:
All samples look pretty good. Oddly, the 260/280 ratios are absolutely perfect, despite the 260/280 ratios from each individual sample being less than stellar (see yesterday's EtOH precipiation). Also of note is that the concentrations for all three samples are extremely close, reflecting the accuracy of the NanoDrop readings of each individual sample used for the pool as well as my pipetting. :)
RNA was stored @ -80C in "Sam's RNA Box #1."
Recovered ~50uL from each sample which means each pool yielded ~3.85ug of RNA after DNase treatment. Will proceed with making cDNA from these three pools. In the interest of time (and the failure of our Opticon), I will not verify that these do NOT still contain gDNA (and, it's pretty unlikely that they do).
20100907
EtOH Precipitation - Hard Clam RNA from earlier today
RNA was mixed with 0.1 vols of 3M NAOAc (pH = 5.2) and 2.5 vols of 100% EtOH, vortexed and incubated @ -20C for 30mins. RNA was pelleted @ 16,000g, 30mins, 4C. Supe was discarded and pellet was washed with 1mL 70% EtOH. RNA was pelleted @ 16,000g, 15mins, 4C. This was step was repeated a second time. Supe was discarded, the RNA was resuspended in 50uL of 0.1% DEPC-H2O, spec'd and stored @ -80C:
Results:
Interestingly, precipitating the samples vastly improved the 260/230 ratios. However, the 260/280 ratios DECREASED for all samples except one (CA 1). Not really thrilled about this fact, nor am I sure why this would happen.
RNA was stored @ -80C in "Sam's RNA Box #1."
RNA Isolation - Hard Clam Tissues Rec'd from Rutgers on 20100820
RNA was isolated from the following samples using TriReagent, according to protocol:
MAX 1, 2, & 3
CA 1, 2, & 3
MA 1, 2, & 3
Samples were resuspended in 50uL of 0.1% DEPC-H2O and spec'd:
Results:
260/280 ratios are decent for most of the samples, with MAX1 and MAX 2 being the exception. Both of these samples also have very poor 260/230 ratios. Out of curiosity, I will EtOH precipitate all samples to see if I can improve both the 260/280 and 260/230 ratios.
20100902
Package - Hard Clam Samples from MBL
Rec'd package from Scott Lindell @ MBL containing 65 screw cap tubes in a white microtube rack. All tubes are tissues in RNA Later (presumably). One sample (MA4-5) was lost during a brief centrifugation to get tissue sample unstuck from top of tube and in to RNA Later solution. The head of the tube snapped off and the entire tube/sample was obliterated in the rotor. Also, it appears as though all the tubes leaked RNA Later solution during transport. Samples were temporarily stored @ 4C and will be catalogued/transferred to -80C.
20100831
Hazardous Waste Collection Request
Collected hazardous waste and submitted
collection request (PDF).
20100820
Package - Hard Clam Samples from Rutgers
Rec'd package of hard clam samples from Emily @ Rutgers on wet ice. Package contained numerous 1.5mL snap cap tubes separated in to groups in zip lock bags. Stored temporarily @ 4C. Will catalog and then store @ -80C.
Three documents included with package:
Note from Emily
Sample info, pg. #1
Sample info, pg. #2
20100709
qPCR - HpaII/MspI Digests from earlier today
qPCR plate layout/setup is here.
Results:
Restriction Digests - Various gigas gDNA from earlier today
Digest master mixes are here. Digests were incubated @ 37C for 2hrs. and then heat inactivated @ 80C for 20mins.
gDNA Isolation - Various gigas samples (continued from yesterday)
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 300uL 8mM NaOH (made 7/9/10 SJW).
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. Used a sample with 8mM NaOH and 1M HEPES as a blank to match the pH = 8.0 of the samples.
Results:
260/280 ratios look good for all samples. Most of the samples have mediocre 260/230 ratios. Yields are excellent for all samples.
20100708
gDNA Isolation - Various gigas samples
Placed ~20mg fragments of tissue in 250uL DNAzol. Added 1.35uL of Proteinase K (Fermentas; 18.5mg/mL) to reach a final concentration of 100ug/mL. Incubated RT, O/N, end-over-end rotation. Will complete DNA isolation tomorrow.
Sample List:
Vt Gigas Live #3 Gill 24E (from
20080828; Tatyana's notebook)
Gigas Control #2 Gill 24E (from
20080828; Tatyana's notebook)
NB-1209-10 (RNA Later)
SB-1209-14 (RNA Later)
WB-1209-09 (RNA Later)
0629 gill 5aza
0629 gonad 5aza
0629 mantle 5aza
MeDIP - SB/WB Fragmented gDNA EtOH precipitation (continued from 20100702)
Finished EtOH precipitation of MeDIP gDNA. Samples were pelleted 16,000g, 4C, 30mins. Supe was discarded. Washed with 1mL 70% EtOH, pelleted 16,000g, 4C, 15mins. Supe discarded. MeDIP DNA was resuspended in 100uL of TE (pH = 8.5). Wash samples, containing unmethylated DNA, were resuspended/combined in a total of 100uL TE (pH = 8.5). Samples were spec'd:
Results:
R37: MeDIP DNA = 1.393ug recovery. This is ~13% of the input total gDNA (11.25ug) and is ~28% of the total DNA recovered in the procedure (4.935ug). Unmethylated DNA = 3.542ug total recovery. This is ~31% of the input total gDNA (11.25ug) and is ~72% of the total DNA recovered in the procedure (4.935ug). Total DNA recovery = ~44%.
R51: MeDIP DNA = 1.256ug recovery. This is ~14% of the input total gDNA (8.75ug) and is ~23% of the total DNA recovered in the procedure (5.462ug). Unmethylated DNA = 4.206ug total recovery. This is ~48% of the input total gDNA (8.75ug) and is ~77% of the total DNA recovered in the procedure (5.462ug). Total DNA recovery = ~62%.
There definitely seemed to be a high degree of salt carryover from the procedure, despite the phenol:chloroform treatment and EtOH precipitation. As such, I believe this is the reason that the 260/230 ratios are so out of whack. Possibly explains why the 260/280 ratios for the MeDIP DNA are so high, too?
These results demonstrate what we can expect to recover from this procedure, as well as how much DNA gets lost during processing. MeDIP DNA and unmethylated DNA were stored @ -20C.
20100702
MeDIP - SB/WB Fragmented gDNA (continued from yesterday)
Continued MeDIP process from yesterday. Protein A/G beads were pelleted XXXXXXXXX, supe transferred to clean tube. Beads were washed 3x in the following fashion, each wash saved to retain unmethylated DNA:
1.
Samples were phenol:chloroform extracted and EtOH precipitated:
1. Added equal volume of phenol:chloroform:IAA, vortexed, spun @ 12,500g, 5mins, 4C.
2. Transferred aqueous phase to clean tube.
3. Added equal volume of chloroform, vortexed, spun @ 12,500g, 5mins, 4C.
4. Transferred aqueous phase to clean tube.
5. Added 0.1 vols 3M NaOAc (pH=5.2), 2.5 vols of 100% EtOH, mixed and stored @ -20C over the weekend.
Will finish precipitation next week and quantify recovery.
Restriction Digests - Various gigas gDNAs of Mac's
Performed restriction digests. Made dilutions of all DNAs involved of 25ng/uL. Made enough for a total of 9 digests could be performed on each DNA. This allowed using 10uL of each DNA for each rxn, more mileage out of the lowest concentration sample (R37-01), and allowed for the use of master mixes when preparing the digests.
All calculations/dilutions/master mixes can be seen here. Each DNA was digested individually with HpaII, MspI and undigested. Incubated the digests 4hrs @ 37C. After digestion, performed an EtOH precipitation. Added 0.1 vols of 3M NaOAc (pH=5.2), then 2.5 vols of 100% EtOH. Mixed by inversion and incubated 30mins @ -20C. Pelleted DNA 16,000g, 30mins @ 4C. Discarded supe. Washed pellets with 1mL 70% EtOH. Pelleted DNA 16,000g, 15mins, 4C. Discarded supe. Resuspended DNA in 10uL PCR H2O and spec'd.
Results:
Well, the recovery of DNA is very low. The best recovery is ~50% while the worst is around ~1%.
I did not proceed with the intended qPCR due to the low yields and the fact that I don't know if we've previously tested how sensitive our assay(s) our for our target genes. Will discuss with Steven/Mac next week.
20100701
MeDIP - SB/WB Fragmented gDNA (continued from yesterday)
Continued MeDIP process from yesterday. Added 20uL of Protein A/G Plus Agarose (Santa Cruz Biotech) beads to each sample and continued incubation with rotation @ 4C for 2hrs. Pelleted the Protein A/G beads 3300g, 2mins, 4C.
Removed and saved supe (to retain unmethylated DNA). Washed beads with 1mL 1x MeDIP Buffer. Repeated two more times. Saved supe after each wash.
Resuspended beads in 250uL MeDIP Digestion Buffer (50mM Tris-HCl, pH=8.0, 10mM EDTA, pH=8.0, 0.5% SDS). Added 75ug of Proteinase K. Incubated 20hrs @ RT with end-over-end rotation.
*Note*: The protocol we have says to incubate the Proteinase K digest @ 55C. However, we don't have a means to do so, since we need a rocker/rotator to keep the agarose beads in suspension. According to various sources, Proteinase K retains >80% of it's enzymatic activity between 20C-50C. So, I've allowed the digest to run longer (24hrs) than recommended (O/N).