20221027 RNA DNA extractions from E5 project

DNA/RNA extractions from E5 project

Re-Extractions from Acropora from the fourth timepoint plus testing two of Zoe’s POR samples

Extraction Date: October 27, 2022

Samples

Tube number Timepoint Species Colony ID Coll date Site
864 November Acropora ACR-267 20201030 2
866 November Acropora ACR-244 20201030 2
872 November Acropora ACR-228 20201030 2
896 November Acropora ACR-234 20201030 2
912 November Acropora ACR-225 20201030 2
920 November Acropora ACR-265 20201030 2
930 November Acropora ACR-237 20201030 2
932 November Acropora ACR-229 20201030 2
936 November Acropora ACR-256 20201030 2
35 January Acropora ACR-223 20200103 2
Z1 NA Porites NA NA NA
Z2 NA Porites NA NA NA

20221027_samples.jpg

Extraction notes

  • For my samples I took 300ul the RNA/DNA shield and added it to a new 1.5ml tube containing 15ul of ProK, and 30ul of ProK digestion buffer
  • For Zoe’s samples I took out 150ul of sample and added it to a new 1.5ml tube containing 150ul of RNA/DNA shield and 15ul of ProK, and 30ul of ProK digestion buffer
  • Samples sat for 15 minutes at room temperate
  • Spun down samples for 3 minutes at 9000 rcf and then transfer the supernatant to new tube without disturbing the pellet
  • All subsequent spins were done for 1 minute or 2.30 minutes
  • Did two washes with 700ul of wash buffer for both the DNA and RNA
  • Then followed the protocol as described in protocol

Qubit

  • Used Broad range dsDNA and RNA Qubit Protocol
  • All samples read twice, standard only read once

DNA

Tube number RFU DNA 1 (ng/uL) DNA 2 (ng/uL) Average
Standard 1 208.79      
Standard 2 24699.59      
864   42.2 42.4 42.3
866   23.8 23.0 23.4
872   40.0 39.4 39.7
896   20.4 19.9 20.15
912   9.9 9.42 9.66
920   8.42 8.38 8.4
930   17.6 17.1 17.35
932   63.2 62.8 63.0
936   70.0 69.8 69.9
35   26.6 26.2 26.4
Z1   ND ND ND
Z2   ND ND ND

RNA

Tube number RFU RNA 1 (ng/uL) RNA 2 (ng/uL) Average
Standard 1 417.40      
Standard 2 9917.82      
864   15.0 14.2 14.6
866   15.4 15.0 15.2
872   17.8 18.2 18.0
896   14.6 16.0 15.3
912   13.2 13.6 13.4
920   19.2 19.0 19.1
930   12.4 12.2 12.3
932   11.8 12.0 11.9
936   ND ND ND
35   11.0 10.8 10.9
Z1   ND ND ND
Z2   ND ND ND

Tape Station

  • Samples not analyzed

Gel

  • Modified from this protocol
  • Added 0.75g of agarose and 50ml of 1x TAE to flask and microwaved for 45 seconds. This makes a 1.5% gel
  • Once cool enough to touch added 2ul of gel green stain
  • Swirled and poured into gel mould with comb
  • Once solidified, covered with 1X TAE as a running buffer
  • Added 1ul of purple loading dye to each of my QC strip tube samples. I had ~9ul of DNA leftover from QC and ~8ul of RNA
  • Loaded my gel with the DNA first, then skipped a well and then the RNA
  • Ran the gel for 60 minutes at 60 volts 202201027_gel.jpg

Addtional Notes

  • Everything looks like garbage :(
Written on October 27, 2022