Pocillopora Samples from Danielle mtORF Amplification and Sanger Sequencing Prep Processing

mtORF Amplification and Sanger Sequencing Prep for Danielle’s Pocillopora Samples

Goal: Amplify Danielle’s 32 samples, with 3 control reactions for mtORF. Then dilute them properly and send them to the GSC for Sanger sequencing. Major Results: Amplifications were really great this time, there was one clear band at ~1000bp which is exactly what we’re supposed to see. It was interesting that rows of the plate had more similar post-PCR concentrations to each other than random samples, even though I tried to dilute all the samples before hand to close to the same concentration.
Major Takeaways: The protocol seems to be working great when scaled up to more samples than the test. I thought about if it would be better to increase the elution volume from the beads so you don’t have to dilute before preparing to sequence, but it would be not good if the concentration then became too low to pipette 25ng in 10ul, and because the PCR concentrations were more variable than I thought they would, I am not decided on if increasing the elution volume from 50ul to like 70ul is a good idea or not. You can always dilute but concentrating is a hassle.

Process

Followed the mtORF amplification protocol exactly. See that for in depth details for this protocol

DNA Dilutions 2020-08-27

  • Arranged samples in a 96 well plate randomly with 3 spots for control reactions
  • Allocated DNA for 10ng/ul in 10ul of nuclease free water, so 100ng of DNA. If samples had concentrations below 10ng/ul to start with, just 10ul of those samples was added to each well

dilplate

mtORF Amplifications 2020-08-27

  • 32 samples plus three negative controls is 35 reactions, use an additional 3 for error
  • Made a master mix for 38 samples and 3 reactions each:
    • 1900ul Phusion master mix
    • 49.4ul FatP6.1 10uM Primer
    • 49.4ul RORF 10uM Primer
    • 1672ul nuclease free water
  • Added 97ul of master mix into 35 wells in a new plate
  • Used a multichannel to add 3ul of DNA from the dilution plate in to the same orientation wells in the plate with the master mix
  • Covered plate and vortexed and spun down
  • Separated plate out 2 times into 3 separated reaction mixes each with 33ul
  • Covered plates, spun down, and placed in three thermocyclers FatP6.1 RORF program

1X Bead Cleanup and Quantification 2020-08-28

  • Combined triplicate reactions back together
  • Added 1X (100ul) beads to each well
  • Followed bead cleanup protocol
  • Resuspended and eluted DNA in 50ul ultra-pure water and removed into a new plate (same orientation)
  • dsDNA broad range Qubit assay for 35 samples (n# 40)
Sample.ID Qubit reading 1 (ng/ul) Qubit reading 2 (ng/ul) average DNA (ng/ul)
C22 29.6 30.2 29.9
E10 30 30 30
E2 27 26.6 26.8
C19 25.8 25.8 25.8
C25 27 26.8 26.9
C29 22 21.8 21.9
C21 25.2 25.2 25.2
E4 22.2 22 22.1
E12 28 28.2 28.1
E1 23 22.4 22.7
C27 21.8 22 21.9
E6 23 23.2 23.1
E8 21.8 21.8 21.8
C20 23 23.2 23.1
C28 21.6 21.6 21.6
E9 23.2 23.4 23.3
E14 17.7 17.7 17.7
E15 17.5 17.6 17.55
C17 21 21 21
E3 21.8 21.8 21.8
C30 18.4 18.4 18.4
C24 19.3 19.4 19.35
E16 39.4 39.8 39.6
C31 35.2 35.4 35.3
E7 42 42.4 42.2
C23 38 38.4 38.2
E11 44.2 44.4 44.3
E13 45.4 45.6 45.5
C18 42.8 43.2 43
E5 46 45.6 45.8
C32 38 38.2 38.1
C26 37.8 37.8 37.8

1% Gel To Confirm Bands 2020-09-01

  • Made a 1% gel in the medium gel box gel

  • One band at ~1000bp, exactly right! No contamination, only imaging artifacts

Dilution and Sequencing Prep 2020-09-02

  • Diluted each DNA amplification by 1:5 (2ul of DNA and 10ul of ultra-pure water)
  • Created plate with 25ng of DNA for each sample and ultra-pure water up to 10ul 25ng
  • Added 2ul of 3.2uM RORF primer to each well
  • Covered, vortexed, spun down plate
  • Brought upstairs for sequencing the next day

Spreadsheet for Sequencing

Sample ID (lab) Sample IDa (GSC) Well (GSC use only) Template Typeb A. Template Size (bases) B. Template Stock Conc. (ng/µl) C. PCR template: ng needed = ((A ÷ 100) x 1.25) x 2 D. PCR template: Volume = (C ÷ B) µl F. Volume PCR-H20 needed (10 minus D or E) µl G. Volume primer needed 1 µl per reaction
C22 HPD1   PCR 1000 5.98 25 4.18 5.82 2
E10 HPD2   PCR 1000 6 25 4.17 5.83 2
E2 HPD3   PCR 1000 5.36 25 4.66 5.34 2
C19 HPD4   PCR 1000 5.16 25 4.84 5.16 2
C25 HPD5   PCR 1000 5.38 25 4.65 5.35 2
C29 HPD6   PCR 1000 4.38 25 5.71 4.29 2
C21 HPD7   PCR 1000 5.04 25 4.96 5.04 2
E4 HPD8   PCR 1000 4.42 25 5.66 4.34 2
E12 HPD9   PCR 1000 5.62 25 4.45 5.55 2
E1 HPD10   PCR 1000 4.54 25 5.51 4.49 2
C27 HPD11   PCR 1000 4.38 25 5.71 4.29 2
E6 HPD12   PCR 1000 4.62 25 5.41 4.59 2
E8 HPD13   PCR 1000 4.36 25 5.73 4.27 2
C20 HPD14   PCR 1000 4.62 25 5.41 4.59 2
C28 HPD15   PCR 1000 4.32 25 5.79 4.21 2
E9 HPD16   PCR 1000 4.66 25 5.36 4.64 2
E14 HPD17   PCR 1000 3.54 25 7.06 2.94 2
E15 HPD18   PCR 1000 3.51 25 7.12 2.88 2
C17 HPD19   PCR 1000 4.2 25 5.95 4.05 2
E3 HPD20   PCR 1000 4.36 25 5.73 4.27 2
C30 HPD21   PCR 1000 3.68 25 6.79 3.21 2
C24 HPD22   PCR 1000 3.87 25 6.46 3.54 2
E16 HPD23   PCR 1000 7.92 25 3.16 6.84 2
C31 HPD24   PCR 1000 7.06 25 3.54 6.46 2
E7 HPD25   PCR 1000 8.44 25 2.96 7.04 2
C23 HPD26   PCR 1000 7.64 25 3.27 6.73 2
E11 HPD27   PCR 1000 8.86 25 2.82 7.18 2
E13 HPD28   PCR 1000 9.1 25 2.75 7.25 2
C18 HPD29   PCR 1000 8.6 25 2.91 7.09 2
E5 HPD30   PCR 1000 9.16 25 2.73 7.27 2
C32 HPD31   PCR 1000 7.62 25 3.28 6.72 2
C26 HPD32   PCR 1000 7.56 25 3.31 6.69 2
Written on August 27, 2020