2022-12-02 Barott and Brown Pdam RNA Plate Prep

Prep and submit RNA for Brown/Barott Pdam Project, December 2, 2022

Dilute extracted RNA to 15 ug/uL in 30 uL and consolidate into one plate for RNAseq. This plate prep guide is based off of Dr. Kevin Wong’s protocol/example here.

Samples

Extraction Order Site Number Sample # Extraction Date RNA_QBIT_AVG Amount of RNA Amount of Water Sample # Plate Well Plate Order
43 RF 17 RF17B 20221111 34.4 13.08 16.92 RF17B A1 1
35 RS 6 RS6A 20221111 36.1 12.47 17.53 RS6A B1 2
55 RS 15 RS15D 20221118 23.6 19.07 10.93 RS15D C1 3
45 RF 19 RF19C 20221111 28.7 15.68 14.32 RF19C D1 4
58 RF 15 RF15D 20221118 45.3 9.93 20.07 RF15D E1 5
56 RF 13 RF13B 20221118 46 9.78 20.22 RF13B F1 6
22 RS 11 RS11B 20221108 23.05 19.52 10.48 RS11B G1 7
38 RS 11 RS11D 20221111 34.9 12.89 17.11 RS11D H1 8
31 RF 20 RF20B 20221108 23.3 19.31 10.69 RF20B A2 9
50 RS 2 RS2C 20221118 24.8 18.15 11.85 RS2C B2 10
30 RF 18 RF18B 20221108 27.3 16.48 13.52 RF18B C2 11
59 RF 22 RF22B 20221118 35.7 12.61 17.39 RF22B D2 12
16 RF 24 RF24B 20221102 25.8 17.44 12.56 RF24B E2 13
21 RS 9 RS9C 20221108 45 10.00 20.00 RS9C F2 14
8 RS 8 RS8B 20221102 24 18.75 11.25 RS8B G2 15
27 RF 14 RF14C 20221108 18.8 23.94 6.06 RD14C H2 16
60 RF 25 RF25A 20221118 59.3 7.59 22.41 RF25A A3 17
28 RF 16 RF16C 20221108 26.5 16.98 13.02 RF16C B3 18
37 RS 9 RS9A 20221111 22.1 20.36 9.64 RS9A C3 19
29 RF 17 RF17D 20221108 34.9 12.89 17.11 RF17D D3 20
51 RS 3 RS3B 20221118 38.4 11.72 18.28 RS3B E3 21
20 RS 7 RS7C 20221108 20.5 21.95 8.05 RS7C F3 22
5 RS 1 RS1B 20221102 22 20.45 9.55 RS1B G3 23
14 RF 19 RF19B 20221102 25.5 17.65 12.35 RF19B H3 24
25 RS 15 RS15B 20221108 19.7 22.84 7.16 RS15B A4 25
39 RS 12 RS12A 20221111 57.6 7.81 22.19 RS12A B4 26
23 RS 12 RS12C 20221108 24.6 18.29 11.71 RS12C C4 27
41 RS 14 RS14B 20221111 37.8 11.90 18.10 RS14B D4 28
48 RF 23 RF23C 20221111 35.5 12.68 17.32 RF23C E4 29
54 RS 14 RS14C 20221118 37.4 12.03 17.97 RS14C F4 30
34 RF 25 RF25C 20221108 28 16.07 13.93 RF25C G4 31
33 RF 24 RF24D 20221108 23.4 19.23 10.77 RF24D H4 32
26 RF 13 RF13D 20221108 32.9 13.68 16.32 RF13D A5 33
44 RF 18 RF18D 20221111 36.9 12.20 17.80 RF18D B5 34
36 RS 8 RS8C 20221111 51.5 8.74 21.26 RS8C C5 35
46 RF 20 RF20C 20221111 42.7 10.54 19.46 RF20C D5 36
57 RF 14 RF14B 20221118 65.3 6.89 23.11 RF14B E5 37
19 RS 3 RS3D 20221108 24.3 18.52 11.48 RS3D F5 38
42 RF 16 RF16A 20221111 43.2 10.42 19.58 RF16A G5 39
40 RS 13 RS13C 20221111 46.5 9.68 20.32 RS13C H5 40
53 RS 7 RS7B 20221118 37.8 11.90 18.10 RS7B A6 41
24 RS 13 RS13A 20221108 42 10.71 19.29 RS13A B6 42
3 RF 15 RF15B 20221027 15.3 29.41 0.59 RF15B C6 43
49 RS 2 RS2B 20221118 30 15.00 15.00 RS2B D6 44
52 RS 6 RS6D 20221118 18.9 23.81 6.19 RS6D E6 45
47 RF 23 RF23A 20221111 47.4 9.49 20.51 RF23A F6 46
32 RF 22 RF22C 20221108 29.2 15.41 14.59 RF22C G6 47
17 RS 1 RS1C 20221108 18.4 24.46 5.54 RS1C H6 48

Plate Map

2022-12-02-plate-map-samples.JPG

Protocol

1) Remove samples from the -80 ºC freezer and place on a tube rack in an ice bucket to thaw.

2) Fill each well of the 96-well PCR plate with the corresponding diluent volume (I used the RNAse/DNAse free water included in the Zymo kit used for extracting the RNA).

2022-12-02-plate-map-dilutent-amt.JPG

3) Place plate onto a block frozen at -80 ºC.

4) Once samples are thawed, spun down, and pipette mixed the sample before adding the appropriate volume into the corresponding well.

2022-12-02-plate-map-RNA-amt.JPG

5) Seal plate with aluminum foil tape and seal each well. Write on the label and the side of the plate with the following information:

- Date: 12/6/22
- Name: Kristen Brown
- Project: Heron Island Pdam
- Job Number: JA22512

6) Place remaining samples and plate back into the -80 ºC freezer.

Written on December 2, 2022