Lab entry 11

Our next step for this lab was to do DD-RADSeq. First, we double digested our DNA samples. To do this, we followed the following steps:

  1. Double digested 100-1000 ng of high quality genomic DNA with selected restriction enzymes, using a digestion buffer appropriate for both enzymes.
  2. Then, I placed 6 uL of each sample’s DNA in the well of a PCR tube, storing it on ice.
  3. Then we prepared the master mix using the following measurements to make 130% excess of master mix 1:
    Master Mix Rxn: 1 Rxns: 11
    CutSmart buffer 10x .9 uL 9.9 uL
    EcoRI-HF enzyme .28 uL 3.08 uL
    MSPI enzyme .12 uL 1.32 uL
    Pure H2O 1.7 uL 18.7 uL
    Master Mix Total: 3 ul 33 ul
  4. We mixed it well, centrifuged it and stored it on ice.
  5. Then, we added 3uL of MM1 to each DNA sample
  6. We sealed the samples, vortexed, centrifuged, and incubated them at 37 degrees Celsius for 8 hours.

My samples were labeled #3 (SCHO002-26) and #4 (DIRA006-11).

Then, we did an adapter ligation. We did this using the following steps:

  1. First, we thawed the working stock EcoRI and Mspl adapters previously made as follows:
    PAUL LAB ID Adapter name
    Eco_2 AACCA_EcoRI
    Eco_3 CGATC_ EcoRI
    Eco_4 TCGAT_ EcoRI
    Eco_5 TGCAT_ EcoRI
    Eco_6 CAACC_ EcoRI
    Eco_7 GGTTG_ EcoRI
    Eco_8 AAGGA_ EcoRI
    Eco_9 AGCTA_ EcoRI
    Eco_10 ACACA_ EcoRI
  2. We added 1 uL of the working stock EcoRI adapter directly to the digested DNA.
  3. Then, we made a second master mix, making 130% excess with the following measurements:
Master Mix Rxn: 1 Rxns: 11
CutSmart buffer 10x .4 uL 4.4 uL
ATP (10mM) 1.3 uL 14.3 uL
T4 Ligase .2 uL 2.2 uL
Pure H2O .1 uL 1.1 uL
Universal P2 Mspl adapter 1.0 uL 11.0
Master Mix Total: 3 ul 33 ul

4. We added 3 uL of MM2 to the digested DNA

5. Finally, we sealed, vortexes, centrifuged, and incubated this at 16 degrees Celsius for 6 hours.


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