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:
- Double digested 100-1000 ng of high quality genomic DNA with selected restriction enzymes, using a digestion buffer appropriate for both enzymes.
- Then, I placed 6 uL of each sample’s DNA in the well of a PCR tube, storing it on ice.
- 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
- We mixed it well, centrifuged it and stored it on ice.
- Then, we added 3uL of MM1 to each DNA sample
- 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:
- 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
- We added 1 uL of the working stock EcoRI adapter directly to the digested DNA.
- 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.