Sam’s Notebook: DNA Isolation and Quantification – C.bairdi Hemolymph Pellets in RNAlater

Isolated DNA from the following 23 samples:

  • 6128_112_9
  • 6204_114_9
  • 6141_123_9
  • 6245_126_9
  • 6240_134_9
  • 6260_136_9
  • 6257_138_9
  • 6259_139_9
  • 6258_140_9
  • 6255_143_9
  • 6256_146_9
  • 6265_155_9
  • 6266_156_9
  • 6261_164_9
  • 6120_165_9
  • 6251_167_9
  • 6262_168_9
  • 6243_173_9
  • 6263_179_9
  • 6264_180_9
  • 6200_208_12
  • 6204_252_12
  • 6190_256_12

Isolated DNA using the Quick DNA/RNA Microprep Kit (ZymoResearch; PDF) according to the manufacturer’s protocol for liquids/cells in RNAlater.

  • Used 35uL from each RNAlater/hemocyte slurry.
  • Mixed with equal volume of H2O (35uL).
  • Retained DNA on the Zymo-Spin IC-XM columns for isolation after RNA isolation.
  • DNA was eluted in 15uL H2O

DNA was quantified on the Roberts Lab Qubit 3.0 using the 1x DNA High Sensitivity Assay (Invitrogen), using 2uL of each sample.

Two samples were too concentrated, so those two samples were re-quantified using 1uL of sample.

Sam’s Notebook: RNA Isolation and Quantification – C.bairdi Hemolymph Pellets in RNAlater

TL;DR – Recovered absolutely no RNA from any sample! However, I did recover DNA from each sample.

Isolated RNA from the following 23 hemolymph pellet samples:

  • 6128_112_9
  • 6204_114_9
  • 6141_123_9
  • 6245_126_9
  • 6240_134_9
  • 6260_136_9
  • 6257_138_9
  • 6259_139_9
  • 6258_140_9
  • 6255_143_9
  • 6256_146_9
  • 6265_155_9
  • 6266_156_9
  • 6261_164_9
  • 6120_165_9
  • 6251_167_9
  • 6262_168_9
  • 6243_173_9
  • 6263_179_9
  • 6264_180_9
  • 6200_208_12
  • 6204_252_12
  • 6190_256_12

Isolated RNA using the Quick DNA/RNA Microprep Kit (ZymoResearch; PDF) according to the manufacturer’s protocol for liquids/cells in RNAlater.

  • Used 35uL from each RNAlater/hemocyte slurry.
  • Mixed with equal volume of H2O (35uL).
  • Retained DNA on the Zymo-Spin IC-XM columns for isolation after RNA isolation.
  • Performed on-column DNase step.
  • RNA was eluted in 15uL H2O

RNA was quantified on the Roberts Lab Qubit 3.0 using the RNA High Sensitivity Assay (Invitrogen), using 2uL of each sample.

Grace’s Notebook: Extract RNA from 24 samples – Day 9, 12 infected, 12 uninfected; still need to run on Qubit

Today I extracted RNA from 24 samples. Day 9, 12 infected and 12 uninfected. I did not run them on the qubit yet. Will do later this week.

Samples I used:

FRP trtmnt_tank day infection_status maturity tube_number
6251 NA 9 1 I 167
6263 NA 9 1 I 179
6262 NA 9 1 I 168
6261 NA 9 1 I 164
6256 NA 9 1 I 146
6257 NA 9 1 I 138
6266 NA 9 1 I 156
6255 NA 9 1 I 143
6240 NA 9 1 I 134
6141 NA 9 1 I 123
6128 NA 9 1 I 112
6118 NA 9 1 I 87
6268 NA 9 0 I 97
6260 NA 9 0 M 136
6259 NA 9 0 M 139
6254 NA 9 0 I 69
6253 NA 9 0 I 25
6248 NA 9 0 I 49
6242 NA 9 0 M 39
6238 NA 9 0 M 9
6235 NA 9 0 I 35
6234 NA 9 0 I 2
6232 NA 9 0 M 52
6230 NA 9 0 M 22

Method

Pretty much did everything the same as this post: Oct 25, 2019

New thing: eluted RNA in 15ul of TE

Where are they now?

Extracted RNA are in -80C (Rack 3, Column 2, Row 3)

DNA are in the fridge in FTR 209 – box 2 of C. bairdi DNA.

Remaining hemolymph samples were placed in Box 2 of the Day 9 (green caps) pellet box (Rack 8, Col 2, Row 5).

Next steps:

Run 2ul on Qubit using RNA HS Kit.

These might help with bulking up the pooled for NWGC that now no longer make the cut-off for their requirements after I attempted to concentrate the RNA (see post: 01/10/2020).

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Grace’s Notebook: 6 pooled Crab RNA samples – unsuccessful concentration with Zymo RNA Clean and Concentrator Kit -5

Today I tried to concentrate the 6 pooled crab RNA samples. Long story short, a lot of RNA was lost in the process and the “concetrated” samples are now well below the requirements for NWGC for RNAseq. Details in post. Next steps, I’m going to extract more RNA from samples from Day 9 so we can bulk those pools up if we want to try to send them to NWGC still. Then I’ll extract more RNA from samples that will bulk up the other pools, too.

6 pooled samples

The initial RNA concentrations for the 6 pooled samples are: (post: here)

trtmnt_tank day infection_status total-yield_ng elution_vol vol_for_pool total_vol_ul qubit_vol_ul qubit_sample_ng total_ng_RNA_pool final_sample_vol
NA 9 0 287.3 13 4.977375566 52.65550411 2 21 1063.765586 50.65550411
NA 9 1 305.5 13 4.680851064 57.13842355 2 21.5 1185.476106 55.13842355
cold 12 0 412.1 13 3.470031546 51.61757335 2 23.6 1170.974731 49.61757335
cold 12 1 280.8 13 5.092592593 39.22471819 2 27.2 1012.512335 37.22471819
warm 12 0 380.9 13 3.754266212 40.86453844 2 26.9 1045.456084 38.86453844
warm 12 1 230.1 13 6.214689266 41.00446376 2 26.3 1025.817397 39.00446376

NWGC requirements

They require normalized at least 1.5 micrograms (15000 ng) of RNA. Each sample should be concentrated to 50ng/ul of RNA. Each sample should be eluted (normalized) in at least 30ul of TE.

Attempt at concentrating

Earlier in the week I tried concentrating two test pools that I made. Post: here. I made a mistake because I diluted the test pools before running 2ul on the qubit, so I didn’t have a baseline idea of how much RNA was in the samples.

Today’s attempt

I know the conenctration and volume of all the samples (in table above).

Because there was a sample that had 55.14ul in it, I decided to normalize them all to 60ul by adding RNAse-free water.

Then I went through the protocol as described.

  1. Add 100ul of RNA Binding Buffer to each sample. Mix by pipet. (Current volume now 160ul).
  2. Add 1 volume (160ul) of 100% ethanol. Mix by pipet. Transfer to a Zymo-Spin IC Column (provided). Centrifuge at 10,000 g for 30s. Discard flow-through.
  3. Add 400ul of RNA Prep Buffer to the column. Centrifuge 10,000g for 30s. Discard flow-through.
  4. Add 700ul of RNA Wash Buffer to the column. Centrifuge 10,000g for 2 minutes. Discard flow-through. Transfer column to labeled RNAse-free microcentrifuge tube.
  5. Add 35ul of TE to the column. Centrifuge 10,000g for 2 minutes. RNA is now eluted in the TE.

Qubit results

I ran 2ul of each sample on the qubit using RNA HS Kit.
The concentrations were wayyyy too far below the required 50ng/ul. And each sample lost RNA, some more than others. 😦

Day Infection status Temperature RNA ng/ul in 2ul Total RNA in sample ng (33ul of sample remaining)
9 0 NA 15.7 518.1
9 1 NA 17.6 580.8
12 0 cold 24.9 821.7
12 1 cold 26.0 858
12 0 warm 26.0 858
12 1 warm 24.4 805.2

Next steps

On Tuesday, January 14th I’ll extract RNA from another set of 24. I’ll focus on Day 9 infected and uninfected again, so that if we want to bulk up those 6 pooled samples with more RNA to send to NWGC, we can.

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Sam’s Notebook: DNA Quality Assessment – Agarose Gel and NanoDrop on C.bairdi gDNA

I isolated C.bairdi gDNA yesterday (20200108) and now want to get an idea if it’s any good (i.e. no contaminants, high molecule weight).

I loaded ~100ng (2uL) of the _C.bairdi_ 20102558-2729 gDNA sample, along with two molecular weight markers (see RESULTS below) on a 0.8% agarose, 1x low TAE gel with ethidium bromide. Gel was run for 1.5hrs at 75V.

Sam’s Notebook: Lab Maintenance – Cluster UPS Battery Replacement

Replaced the batteries on one of the APC uninterruptable power supplies (UPS) on our local server cabinet.

Bad battery indicator light:

APC UPS bad battery indicator light

Sam’s Notebook: DNA Isolation and Quantification – C.bairdi gDNA from EtOH Preserved Tissue

I isolated gDNA from ethanol-preserved C.bairdi muscle tissue from sample 20102558-2729 (SPNO-ReferenceNO). This sample was chosen as it had 0 in the SMEAR_result and BCS_PCR_results columns, indicating it should be free of Hematodinium. See the sample spreadsheet linked below for more info.

C.bairdi ethanol-preserved sample sheet from Pam Jensen (GoogleSheets):

I performed four separate isolations using 50mg of tissue. Samples were “ground” * in liquid nitrogen (LN2) with ceramic mortar/pestle and then processed using the E.Z.N.A. Mollusc DNA Kit according to the manufacturer’s protocol, with the following notes/changes:

  • Used ThermoFisher Proteinase K (18mg/mL) instead of that supplied by kit. Kit Proteinase K was moldy (!); the kit is nearly four years old…
  • Lysed tissue for 1hr.
  • Eluted with 200uL for each sample and pooled for final total volume of ~800uL.

Sample was quantified on the Robert Lab Qubit 3.0 using the DNA Broad Range assay, using 1uL of sample.

*The tissue did not powder as one would expect when grinding in liquid nitrogen. Instead, despite all utensils being pre-chilled with LN2 and tissue being actively ground under LN2, the tissue became a frozen paste that adhered to the pestle. I’ve never experienced this before, but I also have not attempted to grind ethanol-preserved tissue in LN2 before.