This week in lab we performed double-digest restriction associated DNA sequencing
Double Digest of Erythranthe guttate samples:
- To double digest 100-1000ng of high quality genomic DNA with selected restriction enzymes and appropriate digestion buffers, we performed the following steps.
- I labeled 8 PCR tubes 1-8, and added 6uL of each of the following samples’ DNA, to store on ice.
- MONO 005
- DIRA 007
- INVR 001
- CHIM 003
- CHIM 002
- PRBM 005
- CHIM 001
- PRBM 004
- 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).
For this next step in the lab, we used samples that had previously undergone the first 6 steps, and were ready for adapter ligation.
- We pulled out the working stock EcoRI and Mspl adapters made previously, to thaw.
- 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.