November 12 – ddRAD-seq + adapter ligation

In this lab, we digested and ligated our DNA samples.


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


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_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.

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.


Leave a Reply

Your email address will not be published.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