These lab extractions follow the Dual DNA & RNA Extraction Protocol with any deviations, exceptions, or notes explained in detail.

1 August 30th, 2023 (ex10)

1.1 Samples

blank-30aug23
mock-30aug23
1-HA2a
1-HH2a
3-HA2a
3-HH2a
3-MA2b
4-LA2a
4-MA2a

1.2 Results

1.3 Notes

2 August 24th, 2023 (ex9)

2.1 Samples

10-Aa
6-Aa

2.2 Results

2.2.1 Nanodrop

DNA
sample_id	nanodrop_dna	260/280	260/230
10-Aa	177.55	1.87	1.02
6-Aa	140.97	1.89	1.84

RNA
sample_id	nanodrop_rna	260/280	260/230
10-Aa	41.71	2.11	2.19
6-Aa	56.00	2.20	2.05

2.2.2 Qubit

DNA BR Assay Qubit data, RunID 2023-08-24_ (JPG Lab Qubit4), Standard 1: 149.25, Standard 2: 11992.34
sample_id	qubit_dna_1	qubit_dna_2	qubit_dna_avg
10-Aa	169	165	167
6-Aa	131	129	130

RNA BR Assay Qubit data, RunID 2023-08-24_ (JPG Lab Qubit4), Standard 1: 577.12, Standard 2: 18016.50
sample_id	qubit_rna_1	qubit_rna_2	qubit_rna_avg
10-Aa	34.2	34.2	34.2
6-Aa	42.6	43.2	42.9

2.3 Notes

Testing modified protocol with a goal of improving RNA purity on some samples that were taken after the acclimation period in the blue flow-through tanks. These samples are ‘snapshots’ of the corals after they were collected from Kaneohe Bay and fragmented to ~3cm nubbins
Modifications incorporated:
- Added 500uL DNA/RNA Shield to bead bashing tube before adding powdered sample
- After adding the powdered sample, I added another 1000uL of DNA/RNA Shield and vortexed with a quick pulse to submerge all of the powdered sample
- ¹Transferred 600uL of supernatent from bead bashing tube to nuclease-free tube after Mortexing, and saved the bead-bashing tube with remaining ~400uL of homogenized tissue in DNA/RNA Shield as backup
- Transferred 350uL of PK Digested sample to a new tube, saved the leftover PK Digested tube sample as backup
- Added a dry spin for 2min after the last wash buffer spin to ensure complete removal of wash buffer
- eluted ²70uL of DNA & 60uL RNA in Hyclone nuclease-free water (expired Oct 2021..?)

¹ Before this extraction, I had been conducted PK Digestion in the bead bashing tube. I got some advice that this could be contaminating the purity of the RNA:

Jerry Yu from Zymo says:

“We are worried about the sample debris remaining in the tube, which should still contain protein. One of the reasons for Proteinase K treatment at this step is to break down protein in the solution to make it less viscous/sticky, so the column can process it without issue. By transferring the mixture out, the treatment could be more efficient.”

² Kind of arbitrary.. trying to hit something between 100 and 50 microliters

3 June 2nd, 2023 (ex8)

3.1 Samples (DNA only)

blank-02jun23
standard-02jun23

3.2 Results

DNA BR Assay Qubit data, RunID 2023-06-02_133251 (Roberts Lab Qubit4), Standard 1: 151.73 , Standard 2: 13780.58
sample_id	qubit_dna_1	qubit_dna_2	qubit_dna_avg
blank-02jun23	too low	too low	too low
standard-02jun23	21.8	22.2	22

3.3 Notes

Started extraction at the Lyse bead bash step, because both blank and standard are liquids I didn’t need to do the mortar & pestle dance on LN2
Added 75uL of blank & standard to their own correspondingly labelled bead bashing tubes
Used 500uL DNA/RNA Shield
PK Digestion in bead-bashing tubes
No ‘dry spin’ to remove wash buffer
DNA only, these samples are for microbiome contamination assesments
eluted DNA in 50uL nuclease-free water

4 May 19th, 2023 (ex7)

4.1 Samples

3-Eb
2-CH1b
2-PA1b
3-CA1b
3-PH1b
4-PH1b

4.2 Results

DNA BR Assay Qubit data, RunID 2023-05-19_154929 (Roberts Lab Qubit4), Standard 1: 142.53, Standard 2: 10581.91
sample_id	qubit_dna_1	qubit_dna_2	qubit_dna_avg
3-Eb	600	586	593
2-CH1b	9.46	9.16	9.31
2-PA1b	358	350	354
3-CA1b	330	318	324
3-PH1b	48.8	47.2	48
4-PH1b	41.4	40.8	41.1

DNA BR Assay Qubit data, RunID 2023-05-19_153341 (Roberts Lab Qubit4), Standard 1: 566.43 , Standard 2: 16060.47
sample_id	qubit_rna_1	qubit_rna_2	qubit_rna_avg
3-Eb	38.6	38.4	38.5
2-CH1b	33	33	33
2-PA1b	74.6	74.6	74.6
3-CA1b	170	170	170
3-PH1b	69.4	69.8	69.6
4-PH1b	81.2	81.2	81.2

4.3 Notes

Added 500uL of DNA/RNA Shield to each bead-bashing tube
3-CA1b got 79uL of DNase 1 Rx Mix… not quite enough (supposed to be 80uL)

5 May 15th, 2023 (ex6)

5.1 Samples

2-Ea
4-Ea
2-CA2a
4-CH1a

5.2 Results

DNA BR Assay Qubit data, *RunID 2023-05-15_144034 (Roberts Lab Qubit4), Standard 1: 136.34, Standard 2: 8862.90*
sample_id	qubit_dna_1	qubit_dna_2	qubit_dna_avg
2-Ea	248	238	243
4-Ea	222	198	210
2-CA2a	398	390	394
4-CH1a	318	310	314

**RNA BR Assay** Qubit data, RunID 2023-05-15_135647 (Roberts Lab Qubit4), Standard 1: 291.81 , Standard 2: 4673
cryo_id	qubit_rna_1	qubit_rna_2	qubit_rna_avg
2-Ea	too low	too low	too low
4-Ea	660	650	655
2-CA2a	218	216	217
4-CH1a	too low	too low	too low

Important

This Qubit RNA run had a suspiciously low standard #2, so I re-ran it on May 17th

**RNA BR Assay** Qubit data, RunID 2023-05-17_ (Roberts Lab Qubit4), Standard 1: 575.33, Standard 2: 16928.23
cryo_id	qubit_rna_1	qubit_rna_2	qubit_rna_avg
2-Ea	448	446	447
4-Ea	238	240	239
2-CA2a	318	320	319
4-CH1a	83.2	83	83.1

5.3 Extraction Notes

Buffers prepped on 5/12/2023
PK Digestion carried out at 55C for 30min in bead bashing tube
Used frozen DNase Rx Mix from 5/12/2023 on 2-Ea
Not enough DNase 1 left, so for samples 2-CA2a, 4-CH1a & 4-Ea ratio of DNase to buffer was 10uL DNase:300uL Buffer… when it should be 20:300
eluted DNA and RNA in 50uL of Zymo DNA/RNA free water
For QA/QC the first dye+buffer working solution for RNA BR Assay showed a calibration error when running Standard 2, so I remade the dye+buffer working solution. However, the Standard 2 (4673) seemed lower than normal to me, so I re-ran the RNA Qubit for these samples on 5/17/2023 and used those values instead of the ones from 5/15/2023

6 May 12th, 2023 (ex5)

6.1 Samples

1-PA2a
1-PH1a
3-CH2a
4-CA1a
4-PA2a
4-PH1a

6.2 Results

DNA BR Assay, RunID 2023-05-12_160004 (Roberts Lab Qubit4), Standard 1: 141.06, Standard 2: 11326.27
sample_id	qubit_dna_1	qubit_dna_2	qubit_dna_avg
1-PA2a	354	346	350
1-PH1a	438	434	436
3-CH2a	280	276	278
4-CA1a	290	284	287
4-PA2a	584	580	582
4-PH1a	17	16.2	16.6

RNA BR Assay, RunID 2023-05-12_154653 (Roberts Lab Qubit4), Standard 1: 163.75 , Standard 2: 1881.87
sample_id	qubit_rna_1	qubit_rna_2	qubit_rna_avg
1-PA2a	768	756	762
1-PH1a	808	816	812
3-CH2a	too high	too high	too high
4-CA1a	too high	too high	too high
4-PA2a	738	726	732
4-PH1a	45.4	43.2	44.3

6.3 Notes

1-PA2a & 4-CA1a had small amounts of starting material, about half of a 2mL cryovial
4-PH1a had an excess of starting material, full to the top of the bead bashing tube with powdered sample
PK Digestion on heat block set at 55C for 30min, after talk with Zymo rep on the phone
Not enough DNase treatment, so I made a second batch… the kit has very little DNase and it sticks to the lid of the vial, making it really hard to get it all, and there is no ‘extra’ provided. Have to be very careful with the DNase

7 May 5th, 2023 (ex4)

7.1 Samples

1-Ea
2-PA1a

7.2 Results

DNA BR Assay Qubit data, RunID (Roberts Lab Qubit4), Standard 1: 150.76 , Standard 2: 12168.92
sample_id	qubit_dna_1	qubit_dna_2	qubit_dna_avg
1-Ea	202	198	200
2-PA1a	6.5	6.3	6.4

RNA BR Assay Qubit data, RunID (Roberts Lab Qubit4), Standard 1: 571.82 , Standard 2: 16550.34
sample_id	qubit_rna_1	qubit_rna_2	qubit_rna_avg
1-Ea	52.6	52.8	52.7
2-PA1a	154	157	155.5

7.3 Notes

2 big scoops each
PK Digestion for 2 hours at room temp in bead bashing tube
eluted in 50uL Zymo DNA/RNA-free water
Mistake… put 50uL of water in the column when it was over the colelction tube, not in the final nuclease-free tube for saving eluted DNA or RNA. A little bit of water dripped through, I transferred the columns to their nuclease-free tubes and centrifuged… but note here some of the eluted water volume was lost to the collection tube

8 May 2nd, 2023 (ex3) ‘hammer time’

8.1 Samples

1-CA2a
1-CH2a
3-PA1a
3-PH1a

8.2 Results

DNA BR Assay Qubit data, RunID 2023-05-03_140636 (Roberts Lab Qubit4), Standard 1: 147.07, Standard 2: 11753.44
sample_id	qubit_dna_1	qubit_dna_2	qubit_dna_avg
1-CA2a	378	370	374
1-CH2a	197	192	194.5
3-PA1a	396	396	396
3-PH1a	3.3	3.2	3.25

RNA BR Assay Qubit data, RunID 2023-05-03_135153 (Roberts Lab Qubit4), Standard 1: 578.31, Standard 2: 17680.05
sample_id	qubit_rna_1	qubit_rna_2	qubit_rna_avg
1-CA2a	97.2	96.4	96.8
1-CH2a	37	36.8	36.9
3-PA1a	102	102	102
3-PH1a	43.3	43.0	43.2

Very pleased with the RNA quantity result here!

8.3 Notes

2 generous scoopula scoops of powered sample
PK Digestion in bead bashing tube for 2 hours at room temp

9 May 1st, 2023 (ex2)

9.1 Samples

2-PH2a
2-CH1a

9.2 Results

**DNA BR Assay** Standard 1: 147.07, Standard 2: 11753.44
cryo_id	qubit_dna_1	qubit_dna_2	qubit_dna_avg
2-PH2a	59.0	58.6	58.8
2-CH1a	too low	too low	too low

DNA Qubit data, Run 2023-05-03_140008

**RNA BR Assay** Standard 1:321.15 , Standard 2: 5118.36
cryo_id	qubit_rna_1	qubit_rna_2	qubit_rna_avg
2-PH2a	77.6	77	77.3
2-CH1a	21.4	20.8	21.1

RNA Qubit data, Run 2023-05-02_102849

9.3 Notes

2-CH1a: 1 scoop³, 2-PH2a: 2 scoops
Mass/volume of starting material is ~0.16g, or ~200-160uL per ‘scoop’ of powdered sample
Used 500uL of DNA/RNA Shield in each 2mL bead-bashing tube
Switched to metal funnel chilled on dry ice, to keep sample cold (explained further in Dual DNA & RNA Extraction Protocol here)
Let proteinase K digestion incubate at room temp for 2 hours directly in bead bashing tube
Added DNA/RNA Lysis buffer directly to bead bashing tube
eluted DNA & RNA in 100uL nuclease-free water

³ Possibly the low input was a reason for the low DNA yeild

10 April 21st, 2023 (ex1)

10.1 Samples

3-CA1a
3-Ea

Samples were randomly selected for processing given constraints explained in this RMarkdown document.

10.2 Results

**DNA BR Assay, RunID 2023-05-02_102849** Standard 1: 147.07, Standard 2: 11753.44
cryo_id	qubit_dna_1	qubit_dna_2	qubit_dna_avg
3-CA1a	72.8	75	73.9
3-Ea	39	38.4	38.7

DNA Qubit was checked on 04-MAY-2023, Run ID 2023-05-02-102849 because we were waiting for the Thermo Qubit DNA Broad Range (BR) Assay Kit to be delivered. Samples were frozen in a -80C freezer between extraction and getting checked by Qubit for quantity

**RNA BR Assay RunID 2023-04-25_110503** Standard 1: 640.61 , Standard 2: 17282.71
cryo_id	qubit_rna_1	qubit_rna_2	qubit_rna_avg
3-CA1a	12.4	14	12.4
3-Ea	too low	too low	too low

RNA Qubit was run by SST and NAH at 1140 on 25-APR-2023, Run ID 2023-04-25-110503 and on 2 separately prepared technical replicates by SST at 1340 on 25-APR-2023, Run ID 2023-04-25-133744

Samples are stored in -80C freezer in respective Eluted-DNA or Eluted-RNA wax freezer boxes.

10.3 Notes

extractions were started by SST and NAH and finished by SST on 21-APR-2023 ::: column-margin :::
3-CA1a pestled 1st, half of the material in the cryo vial was used. Half remains in the freezer.
3-Ea pestled 2nd, all of the material in the cryo vial was used, but half fell on the floor and had to be thrown out.
LN2 evaporates very quickly and had to be replenished multiple times
We did this with two people (NAH to grind, SST to carefully dispense LN2)

Warning

The coral is very hard to pestle and prone to ‘squirting out’ from under the pestle! Learned that it is better to leverage whole body weight above the mortar and work to crush the coral fragment like a hydraulic press. When pestling sample 3-Ea at an angle, half of the fragment shot out of the mortar and ended up on the floor (which was discarded and not used), resulting in less starting material left to use in the mortar. This could be a reason why 3-Ea had ‘out of range, too low’ result for Qubit!

Used sterilized scoopula to transfer the sample powder to its correspondingly labelled bead bashing tube.

For this step we made small disposable funnels from lab weigh-paper and label tape to try and make this easier. ⚠️Unfortunately, this didn’t work very well! ⚠️ Finely ground powder melts fast and the cold from the LN2 means that all the moisture in the lab is condensing on the sample. As soon as it is removed from the mortar by the scoopula it begins to melt , condense water vapor, and turn into a sticky booger! It doesn’t slide nice and dry down the paper funnel. We had to tamp it vigorously to get it into the bead bashing tube.

Tip

Fixed the melting booger problem by getting small metal funnels and metal scoopulas and chilling them on dry-ice, with the goal being to keep the powder cold during the transfer from the mortar to inside the bead bashing tube. This worked really well!

Once the sample powder was in the bead bashing tube, we quickly added 750uL of DNA/RNA Sheild to the bead bashing tube, shook it vigorously & vortexed it to ensure the sample was submersed in DNA/RNA Sheild

Warning

After bead beating tube was very very bubbly! Would suggest centrifuging after the bead-beating homogenization ‘shaker’ step prior to opening tube lid and putting it in the bead bashing tube before the sample in future extractions.

We then set bead-bashing tubes in the Mortexer and let it mix at max speed for 40 mins
Added 30uL of Proteinase-K Digestion Buffer directly to the bead bashing tube
Added 15uL of Proteinase-K directly to the bead bashing tube
Incubated (left to sit) at room temp for 30 minutes
eluted 100ul of DNA in nuclease-free water
eluted 100ul of RNA in nuclease-free water

11 Template

DNA BR Assay Qubit data, RunID (Roberts Lab Qubit4), Standard 1: , Standard 2:
sample_id	qubit_dna_1	qubit_dna_2	qubit_dna_avg

RNA BR Assay Qubit data, RunID (Roberts Lab Qubit4), Standard 1: , Standard 2:
sample_id	qubit_rna_1	qubit_rna_2	qubit_rna_avg

--- title: "Dual DNA & RNA Extractions for Montipora capitata coral" author: "Sarah Tanja" date: '04/25/2023' categories: [lab records] draft: false image: "steel-cold-funnel.png" --- ::: {.callout-note appearance="simple" icon="false"} These lab extractions follow the [Dual DNA & RNA Extraction Protocol](https://sarahtanja.github.io/quarto-blog/posts/dna-rna-extraction-protocol/) with any deviations, exceptions, or notes explained in detail. ::: # August 30th, 2023 (ex10) ## Samples - blank-30aug23 - mock-30aug23 - 1-HA2a - 1-HH2a - 3-HA2a - 3-HH2a - 3-MA2b - 4-LA2a - 4-MA2a ## Results ## Notes # August 24th, 2023 (ex9) ## Samples - 10-Aa - 6-Aa ## Results ### Nanodrop | sample_id | nanodrop_dna | 260/280 | 260/230 | |-----------|--------------|---------|---------| | 10-Aa | 177.55 | 1.87 | 1.02 | | 6-Aa | 140.97 | 1.89 | 1.84 | : DNA | sample_id | nanodrop_rna | 260/280 | 260/230 | |-----------|--------------|---------|---------| | 10-Aa | 41.71 | 2.11 | 2.19 | | 6-Aa | 56.00 | 2.20 | 2.05 | : RNA ### Qubit | sample_id | qubit_dna_1 | qubit_dna_2 | qubit_dna_avg | |-----------|-------------|-------------|---------------| | 10-Aa | 169 | 165 | 167 | | 6-Aa | 131 | 129 | 130 | : DNA BR Assay Qubit data, RunID 2023-08-24\_ (JPG Lab Qubit4), Standard 1: 149.25, Standard 2: 11992.34 | sample_id | qubit_rna_1 | qubit_rna_2 | qubit_rna_avg | |-----------|-------------|-------------|---------------| | 10-Aa | 34.2 | 34.2 | 34.2 | | 6-Aa | 42.6 | 43.2 | 42.9 | : RNA BR Assay Qubit data, RunID 2023-08-24\_ (JPG Lab Qubit4), Standard 1: 577.12, Standard 2: 18016.50 ## Notes - Testing modified protocol with a goal of improving RNA purity on some samples that were taken after the acclimation period in the blue flow-through tanks. These samples are 'snapshots' of the corals after they were collected from Kaneohe Bay and fragmented to \~3cm nubbins - Modifications incorporated: - Added 500uL DNA/RNA Shield to bead bashing tube **before** adding powdered sample - **After** adding the powdered sample, I added another 1000uL of DNA/RNA Shield and vortexed with a quick pulse to submerge all of the powdered sample - [^1]Transferred 600uL of supernatent from bead bashing tube to nuclease-free tube after Mortexing, and saved the bead-bashing tube with remaining \~400uL of homogenized tissue in DNA/RNA Shield as backup - Transferred 350uL of PK Digested sample to a new tube, saved the leftover PK Digested tube sample as backup - Added a dry spin for 2min after the last wash buffer spin to ensure complete removal of wash buffer - eluted [^2]70uL of DNA & 60uL RNA in Hyclone nuclease-free water (expired Oct 2021..?) [^1]: Before this extraction, I had been conducted PK Digestion in the bead bashing tube. I got some advice that this could be contaminating the purity of the RNA: Jerry Yu from Zymo says: > "We are worried about the sample debris remaining in the tube, which should still contain protein. One of the reasons for Proteinase K treatment at this step is to break down protein in the solution to make it less viscous/sticky, so the column can process it without issue. By transferring the mixture out, the treatment could be more efficient." [^2]: Kind of arbitrary.. trying to hit something between 100 and 50 microliters # June 2nd, 2023 (ex8) ## Samples (DNA only) - blank-02jun23 - standard-02jun23 ## Results | sample_id | qubit_dna_1 | qubit_dna_2 | qubit_dna_avg | |------------------|-------------|-------------|---------------| | blank-02jun23 | too low | too low | too low | | standard-02jun23 | 21.8 | 22.2 | 22 | : DNA BR Assay Qubit data, RunID 2023-06-02_133251 (Roberts Lab Qubit4), Standard 1: 151.73 , Standard 2: 13780.58 ## Notes - Started extraction at the Lyse bead bash step, because both blank and standard are liquids I didn't need to do the mortar & pestle dance on LN2 - Added 75uL of blank & standard to their own correspondingly labelled bead bashing tubes - Used 500uL DNA/RNA Shield - PK Digestion in bead-bashing tubes - No 'dry spin' to remove wash buffer - DNA only, these samples are for microbiome contamination assesments - eluted DNA in 50uL nuclease-free water # May 19th, 2023 (ex7) ## Samples - 3-Eb - 2-CH1b - 2-PA1b - 3-CA1b - 3-PH1b - 4-PH1b ## Results | sample_id | qubit_dna_1 | qubit_dna_2 | qubit_dna_avg | |-----------|-------------|-------------|---------------| | 3-Eb | 600 | 586 | 593 | | 2-CH1b | 9.46 | 9.16 | 9.31 | | 2-PA1b | 358 | 350 | 354 | | 3-CA1b | 330 | 318 | 324 | | 3-PH1b | 48.8 | 47.2 | 48 | | 4-PH1b | 41.4 | 40.8 | 41.1 | : DNA BR Assay Qubit data, RunID 2023-05-19_154929 (Roberts Lab Qubit4), Standard 1: 142.53, Standard 2: 10581.91 | sample_id | qubit_rna_1 | qubit_rna_2 | qubit_rna_avg | |-----------|-------------|-------------|---------------| | 3-Eb | 38.6 | 38.4 | 38.5 | | 2-CH1b | 33 | 33 | 33 | | 2-PA1b | 74.6 | 74.6 | 74.6 | | 3-CA1b | 170 | 170 | 170 | | 3-PH1b | 69.4 | 69.8 | 69.6 | | 4-PH1b | 81.2 | 81.2 | 81.2 | : DNA BR Assay Qubit data, RunID 2023-05-19_153341 (Roberts Lab Qubit4), Standard 1: 566.43 , Standard 2: 16060.47 ## Notes - Added 500uL of DNA/RNA Shield to each bead-bashing tube - 3-CA1b got 79uL of DNase 1 Rx Mix... not quite enough (supposed to be 80uL) # May 15th, 2023 (ex6) ## Samples - 2-Ea - 4-Ea - 2-CA2a - 4-CH1a ## Results | sample_id | qubit_dna_1 | qubit_dna_2 | qubit_dna_avg | |-----------|-------------|-------------|---------------| | 2-Ea | 248 | 238 | 243 | | 4-Ea | 222 | 198 | 210 | | 2-CA2a | 398 | 390 | 394 | | 4-CH1a | 318 | 310 | 314 | : DNA BR Assay Qubit data, *RunID 2023-05-15_144034 (Roberts Lab Qubit4), Standard 1: 136.34, Standard 2: 8862.90* | cryo_id | qubit_rna_1 | qubit_rna_2 | qubit_rna_avg | |---------|-------------|-------------|---------------| | 2-Ea | too low | too low | too low | | 4-Ea | 660 | 650 | 655 | | 2-CA2a | 218 | 216 | 217 | | 4-CH1a | too low | too low | too low | : **RNA BR Assay** Qubit data, RunID 2023-05-15_135647 (Roberts Lab Qubit4), Standard 1: 291.81 , Standard 2: 4673 ::: callout-important This Qubit RNA run had a suspiciously low standard #2, so I re-ran it on May 17th ::: | cryo_id | qubit_rna_1 | qubit_rna_2 | qubit_rna_avg | |---------|-------------|-------------|---------------| | 2-Ea | 448 | 446 | 447 | | 4-Ea | 238 | 240 | 239 | | 2-CA2a | 318 | 320 | 319 | | 4-CH1a | 83.2 | 83 | 83.1 | : **RNA BR Assay** Qubit data, RunID 2023-05-17\_ (Roberts Lab Qubit4), Standard 1: 575.33, Standard 2: 16928.23 ## Extraction Notes - Buffers prepped on 5/12/2023 - PK Digestion carried out at 55C for 30min in bead bashing tube - Used frozen DNase Rx Mix from 5/12/2023 on 2-Ea - Not enough DNase 1 left, so for samples 2-CA2a, 4-CH1a & 4-Ea ratio of DNase to buffer was 10uL DNase:300uL Buffer... when it should be 20:300 - eluted DNA and RNA in 50uL of Zymo DNA/RNA free water - For QA/QC the first dye+buffer working solution for RNA BR Assay showed a calibration error when running Standard 2, so I remade the dye+buffer working solution. However, the Standard 2 (4673) seemed lower than normal to me, so I re-ran the RNA Qubit for these samples on 5/17/2023 and used those values instead of the ones from 5/15/2023 # May 12th, 2023 (ex5) ## Samples 1-PA2a\ 1-PH1a\ 3-CH2a\ 4-CA1a\ 4-PA2a\ 4-PH1a ## Results | sample_id | qubit_dna_1 | qubit_dna_2 | qubit_dna_avg | |-----------|-------------|-------------|---------------| | 1-PA2a | 354 | 346 | 350 | | 1-PH1a | 438 | 434 | 436 | | 3-CH2a | 280 | 276 | 278 | | 4-CA1a | 290 | 284 | 287 | | 4-PA2a | 584 | 580 | 582 | | 4-PH1a | 17 | 16.2 | 16.6 | : DNA BR Assay, RunID 2023-05-12_160004 (Roberts Lab Qubit4), Standard 1: 141.06, Standard 2: 11326.27 | sample_id | qubit_rna_1 | qubit_rna_2 | qubit_rna_avg | |-----------|-------------|-------------|---------------| | 1-PA2a | 768 | 756 | 762 | | 1-PH1a | 808 | 816 | 812 | | 3-CH2a | too high | too high | too high | | 4-CA1a | too high | too high | too high | | 4-PA2a | 738 | 726 | 732 | | 4-PH1a | 45.4 | 43.2 | 44.3 | : RNA BR Assay, RunID 2023-05-12_154653 (Roberts Lab Qubit4), Standard 1: 163.75 , Standard 2: 1881.87 ## Notes - 1-PA2a & 4-CA1a had small amounts of starting material, about half of a 2mL cryovial - 4-PH1a had an excess of starting material, full to the top of the bead bashing tube with powdered sample - PK Digestion on heat block set at 55C for 30min, after talk with Zymo rep on the phone - Not enough DNase treatment, so I made a second batch... the kit has very little DNase and it sticks to the lid of the vial, making it really hard to get it all, and there is no 'extra' provided. Have to be very careful with the DNase # May 5th, 2023 (ex4) ## Samples - 1-Ea - 2-PA1a ## Results | sample_id | qubit_dna_1 | qubit_dna_2 | qubit_dna_avg | |-----------|-------------|-------------|---------------| | 1-Ea | 202 | 198 | 200 | | 2-PA1a | 6.5 | 6.3 | 6.4 | : DNA BR Assay Qubit data, RunID (Roberts Lab Qubit4), Standard 1: 150.76 , Standard 2: 12168.92 | sample_id | qubit_rna_1 | qubit_rna_2 | qubit_rna_avg | |-----------|-------------|-------------|---------------| | 1-Ea | 52.6 | 52.8 | 52.7 | | 2-PA1a | 154 | 157 | 155.5 | : RNA BR Assay Qubit data, RunID (Roberts Lab Qubit4), Standard 1: 571.82 , Standard 2: 16550.34 ## Notes - 2 big scoops each - PK Digestion for 2 hours at room temp in bead bashing tube - eluted in 50uL Zymo DNA/RNA-free water - Mistake... put 50uL of water in the column when it was over the colelction tube, not in the final nuclease-free tube for saving eluted DNA or RNA. A little bit of water dripped through, I transferred the columns to their nuclease-free tubes and centrifuged... but note here some of the eluted water volume was lost to the collection tube # May 2nd, 2023 (ex3) 'hammer time' ## Samples - 1-CA2a - 1-CH2a - 3-PA1a - 3-PH1a ## Results | sample_id | qubit_dna_1 | qubit_dna_2 | qubit_dna_avg | |-----------|-------------|-------------|---------------| | 1-CA2a | 378 | 370 | 374 | | 1-CH2a | 197 | 192 | 194.5 | | 3-PA1a | 396 | 396 | 396 | | 3-PH1a | 3.3 | 3.2 | 3.25 | : DNA BR Assay Qubit data, [RunID 2023-05-03_140636](https://github.com/sarahtanja/coral-DNA-RNA-lab-extractions/blob/main/data/QubitData_2023-05-03/QubitData_2023-05-03_14-06-36.csv) (Roberts Lab Qubit4), Standard 1: 147.07, Standard 2: 11753.44 | sample_id | qubit_rna_1 | qubit_rna_2 | qubit_rna_avg | |-----------|-------------|-------------|---------------| | 1-CA2a | 97.2 | 96.4 | 96.8 | | 1-CH2a | 37 | 36.8 | 36.9 | | 3-PA1a | 102 | 102 | 102 | | 3-PH1a | 43.3 | 43.0 | 43.2 | : RNA BR Assay Qubit data, [RunID 2023-05-03_13515](https://github.com/sarahtanja/coral-DNA-RNA-lab-extractions/blob/main/data/QubitData_2023-05-03/QubitData_2023-05-03_13-51-53.csv)3 (Roberts Lab Qubit4), Standard 1: 578.31, Standard 2: 17680.05 ::: callout-success Very pleased with the RNA quantity result here! ::: ## Notes - 2 generous scoopula scoops of powered sample - PK Digestion in bead bashing tube for 2 hours at room temp # May 1st, 2023 (ex2) ## Samples - 2-PH2a - 2-CH1a ## Results | cryo_id | qubit_dna_1 | qubit_dna_2 | qubit_dna_avg | |---------|-------------|-------------|---------------| | 2-PH2a | 59.0 | 58.6 | 58.8 | | 2-CH1a | too low | too low | too low | : **DNA BR Assay** Standard 1: 147.07, Standard 2: 11753.44 - DNA Qubit data, [*Run 2023-05-03_140008*](https://github.com/sarahtanja/coral-DNA-RNA-lab-extractions/blob/main/data/QubitData_2023-05-03/QubitData_2023-05-03_14-06-36.csv) | cryo_id | qubit_rna_1 | qubit_rna_2 | qubit_rna_avg | |---------|-------------|-------------|---------------| | 2-PH2a | 77.6 | 77 | 77.3 | | 2-CH1a | 21.4 | 20.8 | 21.1 | : **RNA BR Assay** Standard 1:321.15 , Standard 2: 5118.36 - RNA Qubit data, [*Run 2023-05-02_102849*](https://github.com/sarahtanja/coral-DNA-RNA-lab-extractions/blob/main/data/QubitData_2023-05-02/QubitData_2023-05-02_10-32-16.csv) ## Notes - 2-CH1a: 1 scoop[^3], 2-PH2a: 2 scoops - Mass/volume of starting material is \~0.16g, or \~200-160uL per 'scoop' of powdered sample - Used 500uL of DNA/RNA Shield in each 2mL bead-bashing tube - Switched to metal funnel chilled on dry ice, to keep sample cold (explained further in Dual DNA & RNA Extraction Protocol [here](http://localhost:6815/posts/dna-rna-extraction-protocol/#arrange-lab-bench-tubes)) - Let proteinase K digestion incubate at room temp for 2 hours directly in bead bashing tube - Added DNA/RNA Lysis buffer directly to bead bashing tube - eluted DNA & RNA in 100uL nuclease-free water [^3]: Possibly the low input was a reason for the low DNA yeild # April 21st, 2023 (ex1) ## Samples - 3-CA1a - 3-Ea Samples were randomly selected for processing given constraints explained in [this RMarkdown document](https://github.com/sarahtanja/coral-DNA-RNA-lab-extractions/blob/main/rand-sample-processing.Rmd). ## Results | cryo_id | qubit_dna_1 | qubit_dna_2 | qubit_dna_avg | |---------|-------------|-------------|---------------| | 3-CA1a | 72.8 | 75 | 73.9 | | 3-Ea | 39 | 38.4 | 38.7 | : **DNA BR Assay, RunID 2023-05-02_102849** Standard 1: 147.07, Standard 2: 11753.44 - DNA Qubit was checked on 04-MAY-2023, [*Run ID 2023-05-02-102849*](https://github.com/sarahtanja/coral-DNA-RNA-lab-extractions/blob/main/data/QubitData_2023-05-02/QubitData_2023-05-02_10-32-16.csv) because we were waiting for the Thermo Qubit DNA Broad Range (BR) Assay Kit to be delivered. Samples were frozen in a -80C freezer between extraction and getting checked by Qubit for quantity | cryo_id | qubit_rna_1 | qubit_rna_2 | qubit_rna_avg | |---------|-------------|-------------|---------------| | 3-CA1a | 12.4 | 14 | 12.4 | | 3-Ea | too low | too low | too low | : **RNA BR Assay RunID 2023-04-25_110503** Standard 1: 640.61 , Standard 2: 17282.71 - RNA Qubit was run by SST and NAH at 1140 on 25-APR-2023, [*Run ID 2023-04-25-110503*](https://github.com/sarahtanja/coral-DNA-RNA-lab-extractions/blob/main/data/QubitData_2023-04-25/QubitData_2023-04-25_13-41-07.csv) and on 2 separately prepared technical replicates by SST at 1340 on 25-APR-2023, [*Run ID 2023-04-25-133744*](https://github.com/sarahtanja/coral-DNA-RNA-lab-extractions/blob/main/data/QubitData_2023-04-25/QubitData_2023-04-25_13-41-07.csv) Samples are stored in -80C freezer in respective `Eluted-DNA` or `Eluted-RNA` wax freezer boxes. ## Notes - extractions were started by SST and NAH and finished by SST on 21-APR-2023 ::: column-margin ![mortar and pestle with cryogloves](PXL_20230421_213402619.jpg){width="3in"} ::: - `3-CA1a` pestled 1st, half of the material in the cryo vial was used. Half remains in the freezer. - `3-Ea` pestled 2nd, all of the material in the cryo vial was used, but half fell on the floor and had to be thrown out. - LN2 evaporates very quickly and had to be replenished multiple times - We did this with two people (NAH to grind, SST to carefully dispense LN2) ::: callout-warning The coral is very hard to pestle and prone to 'squirting out' from under the pestle! Learned that it is better to leverage whole body weight above the mortar and work to crush the coral fragment like a hydraulic press. When pestling sample 3-Ea at an angle, half of the fragment shot out of the mortar and ended up on the floor (which was discarded and not used), resulting in less starting material left to use in the mortar. **This could be a reason why 3-Ea had 'out of range, too low' result for Qubit!** ::: - Used [sterilized](#sterilize) scoopula to transfer the sample powder to its correspondingly labelled [bead bashing tube](https://www.zymoresearch.com/collections/lysis-tubes/products/zr-bashingbead-lysis-tubes-0-1-0-5-mm). For this step we made small disposable funnels from lab weigh-paper and label tape to try and make this easier. ⚠️**Unfortunately, this didn't work very well!** ⚠️ Finely ground powder melts fast and the cold from the LN2 means that all the moisture in the lab is condensing on the sample. As soon as it is removed from the mortar by the scoopula it begins to melt , condense water vapor, and turn into a sticky booger! It doesn't slide nice and dry down the paper funnel. We had to tamp it vigorously to get it into the bead bashing tube. ::: callout-tip Fixed the melting booger problem by getting [small metal funnels](https://www.amazon.com/Yansanido-Stainless-Miniature-Essential-Lipbalms/dp/B086MCNT66/ref=sxts_rp_s_a_1_0?content-id=amzn1.sym.3432eb1a-1558-4445-9430-9bb3e7f7b9b7%3Aamzn1.sym.3432eb1a-1558-4445-9430-9bb3e7f7b9b7&cv_ct_cx=small+metal+funnel&keywords=small+metal+funnel&pd_rd_i=B086MCNT66&pd_rd_r=ea974960-1e0a-42ee-ad24-697dac76e00c&pd_rd_w=qJ6ax&pd_rd_wg=BfUCw&pf_rd_p=3432eb1a-1558-4445-9430-9bb3e7f7b9b7&pf_rd_r=1F9G2RCT07066Q1KP00D&qid=1685747165&sbo=RZvfv%2F%2FHxDF%2BO5021pAnSA%3D%3D&sr=1-1-5985efba-8948-4f09-9122-d605505c9d1e) and metal scoopulas and chilling them on dry-ice, with the goal being to **keep the powder cold during the transfer from the mortar to inside the bead bashing tube**. This worked really well! ::: ::: column-margin ![funnel weigh paper diagram](funnel.png) ::: - Once the sample powder was in the bead bashing tube, we quickly added `750uL` of `DNA/RNA Sheild` to the bead bashing tube, shook it vigorously & vortexed it to ensure the sample was submersed in `DNA/RNA Sheild` ::: callout-warning After bead beating tube was very very bubbly! Would suggest centrifuging after the bead-beating homogenization 'shaker' step prior to opening tube lid and putting it in the bead bashing tube before the sample in future extractions. ::: - We then set bead-bashing tubes in the `Mortexer` and let it mix at max speed for **40 mins** - Added `30uL` of Proteinase-K Digestion Buffer directly to the bead bashing tube - Added `15uL` of Proteinase-K directly to the bead bashing tube - Incubated (left to sit) at room temp for **30 minutes** - eluted 100ul of DNA in nuclease-free water - eluted 100ul of RNA in nuclease-free water ------------------------------------------------------------------------ # Template | sample_id | qubit_dna_1 | qubit_dna_2 | qubit_dna_avg | |-----------|-------------|-------------|---------------| | | | | | | | | | | | | | | | | | | | | : DNA BR Assay Qubit data, RunID (Roberts Lab Qubit4), Standard 1: , Standard 2: | sample_id | qubit_rna_1 | qubit_rna_2 | qubit_rna_avg | |-----------|-------------|-------------|---------------| | | | | | | | | | | | | | | | | | | | | : RNA BR Assay Qubit data, RunID (Roberts Lab Qubit4), Standard 1: , Standard 2: