Ran out 2uL of Clair’es C.gigas female gonad gDNA (from 20140328) and Mac’s C.gigas gonad gDNA (from 20140402) for quality assessment. Both samples had been isolated using Qiagen’s Blood & Tissue DNeasy Kit. 2uL of each sample was run on a 0.8% 1x TBE gel.
Results:
Loading:
Lane 1 – Hyperladder 1 (Bioline)
Lane 2 – Claire’s gDNA
Lane 3 – mac’s gDNA
Both samples show an extremely high amount of smearing. Additionally, both samples have definitive bands that correspond to ~1300bp and ~850bp.
Tried another method of RNA Isolation for comparison with regular TriReagent method.
Used the Direct-zol RNA MiniPrep Kit (Zymo Research) on the following samples stored in RNAlater:
Pelleted samples in RNAlater by spinning 5000g, 10mins @ RT. Removed RNAlater, lysed pellets in 1mL TriReagent. Split each sample equally into two tubes (500uL in each tube). Added equal volumes of 100% ethanol to each tube and vortexed. Transferred samples to spin columns and followe manufacturer’s protocol. Eluted with 25uL of nuclease-free H2O (provided in kit). Spec’d on NanoDrop1000.
Results:
RNA quality is very good (based on 260/280 ratios). This turned out much better than the previous attmpt using the basic TriReagent method. However, the previous attempt (see 20140401) may have been compromised by me being too aggressive when collecting the aqueous phase. Knowing how little sample was present, I may have been overzealous in trying to gather too much of the aqueous phase, leading to the phenol carryover that was evident.
Regardless, these columns seem to do an excellent job of eliminating even salt carryover, as we frequently see high absorbance at 230nm with marine samples; particularly those stored in RNAlater.
Isolated RNA from the following samples (stored in RNAlater):
P18 Control 3/17/14
P10 Filt. Inj. 3/17/14
These were “trial” RNA isolation runs to determine what yields we could expect from samples of this nature.
Both samples had very small tissue/cell pellets. Tubes were spun @ 5000g for 10mins at RT to ensure all cells were pelleted. RNAlater was removed and pellets were lysed using 1000uL of TriReagent, supplemented with 8uL of PolyAcryl carrier. PolyAcryl Carrier was used to enhance RNA recovery from such small starting materials. Remainder of procedure followed manufacturer’s protocol. RNA was resuspended in 20uL of 0.1% DEPC-H2O and spec’d on a NandoDrop1000.
Results:
As can be seen by the absorbance spectrum plots (top image), there is clear phenol contamination (indicated by shift of absorbance peak to 270nm, instead of the peak being at 260nm). Additionally, there’re large peaks at 230nm in each of the two samples, suggesting other contamination (high residual salts, ethanol?). Additionally, the 260/280 ratios are subpar for RNA quality (i.e. <1.9). However, these ratios could be skewed by the the residual phenol present in both samples. I may perform an ethanol precipitation on these just to see if I can get them cleaned up.
Yields for both samples are very promising.
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:
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.
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:
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).
We’ve had some Illumina sequencing issues with Yanouk’s samples, so I ran the samples out on a 0.8% agarose gel to evaluate the levels of degradation. Loaded 2uL of each sample. Did not load equal quantities of gDNA, due to the lack of available gDNA in the samples we submitted for Illumina sequencing. Added 2uL of H2O to samples 37 & 38 in hopes of having sufficient DNA for visualization on the gel.
See Sam’s Notebook 20131004 for sample concentrations.
Results:
First lane is Hyperladder II (Bioline). Highest molecular weight of the ladder is 2kb.
Sample numbers are listed above each lane.
All samples show a significant amount of smearing, but all still have an identifiable high molecular weight band. Will show to Steven and discuss options for re-sequencing.
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.
After speaking with Axa regarding the DNA concentrations, he would like the DNA from the ethanol-fixed tissue to be more concentrated, and he wants them in ddH2O instead of Buffer AE (from the Qiagen DNeasy Kit). So, I preformed a standard ethanol precipitation. Added 0.1 volumes of 3M sodium acetate (pH = 5.2) [15uL], 2.5 volumes of 100% ethanol [412.5uL] and incubated @ -20C over the weekend.
Pelleted DNA by spinning 16,000g, 15mins @ 4C. Discarded supe, washed pellets with 1mL 70% ethanol and re-pelleted the DNA. Performed a second wash with 70% EtOH, pelleted DNA, discarded supe, resuspended DNA in 75uL of NanoPure H2O, and spec’d on NanoDrop1000.
Results:
Will spec when I re-isolate DNA from “fresh” geoduck samples for coordinating Axa’s DNA sequencing project with our potential RNA-seq project. See 20140219.
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:
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.
Isolated gDNA from 6 geoduck siphon samples provided by Brent using the Qiagen DNeasy Spin Kit. Samples were as follows:
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 100uL of Buffer AE and spec’d on a NanoDrop1000.
Results:
The person who needs these samples (Axa) needs at least 25ug of DNA. The two fresh samples (Geoduck 01 and Geoduck 02) yielded more than sufficient quantities of DNA. The Langley (i.e. ethanol-fixed) samples did not yield sufficient DNA and I will need to isolate additional DNA from these samples.