The following links show the statistics provided by MinKNOW for the four DNA sequencing runs I performed two weeks ago. More later…
Another day, another DNA extraction run. This time the results were quite good, which is especially good considering that these samples were from the anemone tentacle crowns and are the samples of interest for further work. A lesser amount of tissue appears to do fine when it is liquid nitrogen ground, which is not surprising. Here are the results (Qiagen DNeasy, 12 hr digest, 50 ul elution, Qubit DNA HS assay with 3ul sample). Samples are labelled “DNA-2″. I ran a gel and the DNA quality was also excellent, some of the best I have seen. Note that realistically remaining volumes are more like 44 ul after what was used for Qubit and gel. But that still gives more than 1 ug total. Also, a second elution with 50 ul was done in a separate tube, so there is more DNA available but not quantified.
|Sample||Vol ul||DNA ng/ul|
I did another round of DNA and RNA extractions yesterday. First I did some RNA extractions on some frozen tissues that consisted of only tentacle crowns, the idea being that these tissues will be the most enriched with symbionts. I used the Qiagen RNeasy kit followed by quantification using the Qubit RNA HS assay, with 1 ul of sample. Many of these preps had RNA concentrations over the detection limit so they will need to be diluted.
Note: sample G3 was extracted on 8/28/18 and quantified the same day. Also note these are labeled “RNA-2″.
|Sample||Vol ul||RNA ng/ul|
Next I wanted to examine the effect of tissue concentration on DNA extractions since my previous round of extractions gave very low yields. I wondered if I had used either too much or too little tissue. These samples were ground in liquid nitrogen and I have less experience extracting DNA from such samples. It is very possible that the ground samples extract much more efficiently and that I was using too much sample. Unfortunately the results are not totally clear, as the initial quantity of ground tissue did not seem to have a major effect on DNA yield. My hypothesis is that the low quantities of tissue may have saturated the columns and therefore the higher quantities of tissue gave similar yields due to the combined effects of more DNA and yet more saturation or clogging of the membranes. The DNA was eluted with a single pass of 50 ul AE buffer and quantified using the Qubit DNA HS kit with 3 ul of sample.
|Sample||Vol ul||DNA ng/ul|
Yesterday and today I did some more DNA and RNA extractions from A. elegantissima tissue using the Qiagen DNeasy and RNeasy kits. I use the Qubit DNA HS and RNA kits to assess quantity. Here are the results:
|Sample||DNA ul||DNA ng/ul||RNA ul||RNA ng/ul|
Continued RNA extractions of Anthopleura elegantissima samples using the Qiagen RNeasy kit. Further information on these samples can be found in this post. Qiashredder columns were again used for sample homogenization, and the Qubit RNA HS assay for quantification. Here are the results:
|Species||Sample||RNA (ng/ul)||Total vol (ul)||Notes|
I did some further RNA extractions of Porites astreoides and Anthopleura elegantissima samples using the Qiagen RNeasy kit. Further information on these samples can be found in the previous post. I again used Qiashredder columns for sample homogenization, and use the Qubit RNA HS assay for quantification. Here are the results:
|Organism||Sample||RNA ng/ul||Sample vol (ul)||Notes|
|A. elegantissima||A4 483||>100||40|
|A. elegantissima||M4 414||<20||40|
|A. elegantissima||H1 400||>100||40|
|A. elegantissima||H6 120||>100||40|
|A. elegantissima||M3 461||>100||40||lowest amount of starting material|
|A. elegantissima||A2 438||>100||40|
I may do some dilutions later.
I just noticed that the last time I posted was about a year ago – wow, time flies! I have primarily been working on RADseq data assemblies (i.e., being a computer jockey) and have not done really any bench work since then, hence the gap.
I’m finally getting into some RNA extractions on both coral and anemone samples. The coral samples are Porites astreoides from the Belize transplant experiment, collected in November 2016. The anemone samples are Anthopleura elegantissima from Natalie Coleman’s OA experiment last summer. Both sets of samples were flash frozen in liquid nitrogen and stored in a -80°C freezer since then. In January, I crushed the majority of these samples to a fine powder under liquid nitrogen using a mortar and pestle. The coral samples ended up being more substantial and I only needed a portion of each, and the crushed powder I obtained weighed approximately 500-700 mg per sample. The anemones samples, on the other hand, were smaller, and I only got about 60-150 mg per sample. Granted, some of the weight of the coral samples is probably skeleton.
For the extractions, I used the Qiagen RNeasy kit, with initial sample homogenization with Qiashredder columns. I used the animal tissues protocol with a final elution volume of 40 µl (single pass). The following are results from the Qubit RNA HS assay:
|Organism||Sample||RNA (ng/ul)||Sample vol (ul)||Notes|
|P. astreoides||Past13 ’16 Home||35.9||40|
|P. astreoides||Past13 ’16 CBC||45.7||40|
|A. elegantissima||H2 End 422||too low||40||Low amount of starting material; thawed slightly|
|A. elegantissima||A2 End 6||too low||40||Low-ish starting amount|
|A. elegantissima||M3 End 468||124||60||Had to be diluted to Qubit|
|A. elegantissima||A1 end 425||71.6||60||Had to be diluted to Qubit|
Overall I am pleased with these results. The only one I find odd and hard to explain is H2 End 422 – this had a relatively low amount of starting material, but I didn’t think it was that low. It may have thawed or been otherwise compromised, but I made no note of this.
1X Phosphate Buffered Saline (PBS Buffer) Recipe
Dissolve in 800ml distilled H2O:
8g of NaCl
0.2g of KCl
1.44g of Na2HPO4
0.24g of KH2PO4
Adjust pH to 7.4 with HCl.
Adjust volume to 1L with distilled H2O.
Sterilize by autoclaving.
Yesterday I completed some re-do PCRs of Symbiodinium cp23S from the branching Porites samples Sanoosh worked on over the past summer. Some of the samples did not amplify at all, so I reattempted PCR of these samples (107, 108, 112, 116). Sample 105 amplified last summer but the sequence was lousy, so I redid that one too. After the first PCR, I obtained 1 ul of the product and diluted it 1:100 in water. I then used 1 ul of this diluted product as the template for a second round of PCR. PCR conditions were the same Sanoosh and I used last summer (based on Santos et al. 2002):
|5X Green Buffer||2.5|
|MgCl2 25 mM||2.5|
|dNTP mix 10 mM||0.6|
|Go Taq 5U/µl||0.2|
|primer 23S1 10 µM||0.5|
|primer 23S2 10 µM||0.5|
|Master Mix volume||24|
Initial denaturing period of 1 min at 95 °C, 35 cycles of 95 °C for 45 s, 55 °C for 45 s, and 72 °C for 1 min, and a final extension period of 7 min.
Samples were then run on a 1% agarose gel for 30 min at 135 volts.
Surprisingly, the first round of PCR amplified samples 107 and 112 (note: two subsamples of each were run; one that was the original extraction diluted (d) and another that was the original cleaned with Zymo OneStep PCR Inhibitor Removal kit (c)). The cleaned samples were the ones that amplified. I believe Sanoosh had tried these cleaned samples with no success.
The second round of PCR produced faint bands for both of the 108 samples. Sample 116 still did not amplify.
I cleaned the samples with the NEB Monarch Kit and shipped them today to Sequetech. I combined the two 108 samples to ensure enough DNA for sequencing.
This is a belated post for some RAD library prep I did the week of January 23rd in the Leache Lab. I followed the same ddRAD/EpiRAD protocol I used in August. Samples included mostly Porites astreoides from the transplant experiment, as well as some geoduck samples from the OA experiment, and a handful of green and brown Anthopleura elegantissima. Sample metadata can be found here. The library prep sheet is here. The TapeStation report is here. Below is the gel image from the TapeStation report showing that the size selection was successful. However, the selection produced fragments with a mean size of 519-550 base pairs, as opposed to the size selection in August which produced ~500 bp fragments. While there will obviously be some overlap between libraries, combining samples from the two libraries may be problematic. This occurred despite identical Pippen Prep settings targeting fragments 415-515 bp. Libraries were submitted to UC Berkeley on 1/31/17 for 100 bp paired-end sequencing on the HiSeq 4000.