Lab 11: DD-RADSeq (Double-digest restriction associated DNA Sequencing)

Allyson Luber

November 12, 2019

DD-RADSeq (Double-digest restriction associated DNA Sequencing) Lab

I. Double Digest : digesting our M. guttatus samples. The objective of this part of the lab was to double digest 100-1000 ng of high quality genomic DNA with selected restriction enzymes, using a digestion buffer appropriate for both enzymes.

  1. Placed 6 μL of each of our sample’s DNA in a PCR tube, then stored it on ice
  2. Prepared Master Mix 1: Recipe for one double-digest reaction = 3.0 μL
  3. Added 3  μL of Master Mix 1 to each M. guttatus DNA sample
  4. We observed that the total reaction volume is now 9  μL.
  5. Next, we sealed the samples, vortexed, centrifuged, and incubated at 37 degrees Celsius for 8 hours on a thermocycler with a heated lid set to 50 degrees Celsius (run DD on BIORAD #11-2 –> DDRAD –> DD)

Master Mix 1 Recipe:

Master Mix: 11 RXN’s (due to the small volumes used and the viscous nature of the glycerol the enzymes are stored in, we made at least a 130% excess of MM1 to accommodate multiple rounds of pipetting)

  • CutSmart Buffer 10X (0.90  μL/1 rxn) : 9.9  μL
  • EcoRI-HF enzyme (0.28  μL/1 rxn): 3.08  μL
  • MSPI enzyme (0.12  μL/ 1 rxn): 1.32  μL
  • Pure H2O (1.70  μL/ 1 rxn): 18.7  μL
  • Master Mix total (3.00  μL/ 1 rxn) : 51.7  μL

Double Digest tubes:

  1. SAMPLE 7: MONO-002 (RICKY)
  2. SAMPLE 15: DIRA-010
  3. SAMPLE 24: LOTR-002 (ELI)
  4. SAMPLE 8: MONO-003
  5. SAMPLE 4: CHIM-004 (ALLYSON)
  6. SAMPLE 16: PRBN-001

I. Adapter Ligation: Performed all steps with samples in ice

  1. I added 1  μL of the working stock EcoRI adapter directly to the digested DNA
  2. Due to the small volumes used and the viscous nature of the glycerol the enzymes were stored in, we made at least a 130% excess of master mix 2 to accommodate multiple rounds of pipetting
  3. Prepared MM2
  4. Added 3  μL of MM2 to the digested DNA
  5. The total reaction volume is now 13 μL. (Dr. Paul did this part: incubate at 16 degrees celsius for 6 hours on a thermocycler with a heated lid set to 50 degrees celsius). After ligation the samples can be stored frozen for a few weeks, if needed.

Ligation tubes

  1. SAMPLE 25: ECO-2
  2. SAMPLE 26: ECO-3
  3. SAMPLE 27: ECO-4
  4. SAMPLE 28: ECO-5
  5. SAMPLE 29: ECO-6
  6. SAMPLE 30: ECO-7
  7. SAMPLE 31: ECO-8
  8. SAMPLE 32: ECO-9

Adapter Ligation Master Mix recipe for RADseq (Master Mix 2): 11rxn’s. Before making this Master Mix, we added 1  μL of the EcoRI adapter assigned to us to each of our digested DNA products. Recipe for one adapter ligation reaction is 3.0  μL

  • CutSmart buffer 10X (0.40 μL/1 rxn): 4.4  μL
  • ATP (10mM) (1.30 μL/1 rxn): 14.3  μL
  • T4 Ligase (0.20  μL/1 rxn): 2.2  μL
  • Pure H2O (0.10  μL/ 1rxn): 1.1
  • Universal P2 MspI adapter (1.00 μL/1  μL): 11  μL
  • Master Mix total (3.00  μL/1 rxn): 33  μL

Leave a Reply

Your email address will not be published. Required fields are marked *