This week in lab we performed double-digest restriction associated DNA sequencing

Double Digest of Erythranthe guttate samples:

  1. To double digest 100-1000ng of high quality genomic DNA with selected restriction enzymes and appropriate digestion buffers, we performed the following steps.
  2. I labeled 8 PCR tubes 1-8, and added 6uL of each of the following samples’ DNA, to store on ice.
    1. MONO 005
    2. DIRA 007
    3. INVR 001
    4. CHIM 003
    5. CHIM 002
    6. PRBM 005
    7. CHIM 001
    8. PRBM 004
  3. We prepared a Master Mix for our table. Due to the small volumes used and the viscous nature of the glycerol the enzymes are stored in, we made 130% excess (enough for 11 reactions) of the following recipe:
  • 0.9 uL CutSmart 10X buffer/rxn   (9.9 uL total)
  • 0.28 uL EcoRI-HF enzyme/rxn.   (3.1 uL total)
  • 0.12 uL MPSI enzyme/rxn.           (1.32 uL total)
  • 1.7 uL pure H2O/rxn                     (18.7 uL total)

4. We stored the Master Mix 1 on ice.

5. We added 3 uL of Master Mix 1 to each sample’s DNA, changing pipette tips with every sample.

6. I sealed, vortexes and centrifuged the samples, and then added them to thermocycler with a heated lid set to 50 degrees Celsius, to incubate at 37 degrees Celsius for 8 hours (ran “DD” on BIORAD #1/2 –> DDRAD –>DD).

 

Adapter Ligation:

For this next step in the lab, we used samples that had previously undergone the first 6 steps, and were ready for adapter ligation.

  1. We pulled out the working stock EcoRI and Mspl adapters made previously, to thaw.
  2. We added 1 uL of the working stock EcoRI adapter directly to the digested DNA. We used different adapters in each sample, as paired below:

Sample 9 – Eco 2

Sample 10 – Eco 3

Sample 11 – Eco 4

Sample 12 – Eco 5

Sample 13 – Eco 6

Sample 14 – Eco 7

Sample 15 – Eco 8

Sample 16 – Eco 9

3. We prepared a Master Mix 2 for our table. Due to the small volumes used and the viscous nature of the glycerol the enzymes are stored in, we made 130% excess (enough for 11 reactions) of the following recipe:

  • 0.4 uL CutSmart 10X buffer/rxn                      (4.4 uL total)
  • 1.3 uL ATP 10mM/rxn                                        (14.3 uL total)
  • 0.2 uL T4 Ligase enzyme/rxn                            (2.2 uL total)
  • 0.7 uL pure H2O/rxn                                             (7.7 uL total)
  • 1.0 uL working stock universal P2 Mspl adapter  (11 uL)

4. I added 3 uL of Master Mix 2 to the digested DNA samples.

5. I sealed the samples, vortexes, centrifuged and added to a thermocycler with a heated lid set to 50 degrees Celsius to incubate at 16 degrees celsius for 6 hours (ran “LIGATE” of BIORAD #1/2 –>DDRAD –>LIGATE).

6. After the run, the samples were stored in a freezer.