In this lab, we digested and ligated our DNA samples.
I. DOUBLE DIGEST
For this part of the lab, we first double digested 100-1000 ng of high quality genomic DNA with restriction enzymes then used a digestion buffer appropriate for the two enzymes. We placed 6 microliter of each sample’s DNA in a PCR plate/tube and stored it on ice.
Then, we made a master mix for around 10-11 samples to make sure we had enough for our 8 reactions. We mixed it well and then stored it on ice.
| CutSmart Buffer 10x | 0.90 microliters | 9.90 microliters |
| EcoRI-HF enzyme | 0.28 microliters | 3.08 microliters |
| MSPI enzyme | 0.12 microliters | 1.32 microliters |
| Pure H2O | 1.70 microliters | 18.7 microliters |
| Master Mix total | 3.00 microliters | 33.0 microliters |
Next, we added 3 microliter of master mix into each sample.
- the total reaction volume at this point was 9 microliter. We sealed the samples, vortexes them, centrifuged, and finally incubated them at 37 degrees Celsius for 8 hours on a thermocycler with a heated lid set to 50 degrees.
Our samples names and numbers from Alec (grad student who collected these samples) are shown below-
| DIRA009 | 14 |
| INVR002 | 22 |
| SCHO002 | 26 |
| DIRA006 | 11 |
| MONO006 | 10 |
| CHIM005 | 5 |
| PRBN002 | 17 |
| TROS002 | 28 |
II. ADAPTER LIGATION
The next part of this lab was the adapter ligation. We thawed the working stock EcoRI and Maple adapters made previously in the Paul Lab. The P1 EcoRI adapters are 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 |
Our table was assigned 17-24 as our sample ID’s so we used Eco Adapters 2-9 respectively- those are shown below as well.
| SAMPLE ID | ECO ADAPTER |
| 1 (17) | 2 |
| 2 (18) | 3 |
| 3 (19) | 4 |
| 4 (20) | 5 |
| 5 (21) | 6 |
| 6 (22) | 7 |
| 7 (23) | 8 |
| 8 (24) | 9 |
We added one microliter of each respective EcoRI adapter to the digested DNA, then made the master mix. Again, we made 11 reactions worth of master mix (130%).
| CutSmart buffer 10x | 0.4 microliters | 4.4 microliters |
| ATP (10mM) | 1.30 microliters | 14.3 microliters |
| T4 ligase | 0.20 microliters | 2.2 microliters |
| Pure H2O | 0.10 microliters | 1.1 microliters |
| Universal P2 MspI adapter | 1.00 microliters | 11.0 microliters |
| Master Mix Total | 3.00 microliters | 33.0 microliters |
We added three microliters of this second master mix to the digested DNA
- The total reaction volume is now 13 microliter. Samples were sealed, vortexed, centrifuged, and incubated at 16 degrees Celsius for 6 hours on a thermocycler with a heated lid set to 50 degrees Celsius.
