Double Digestion and Adapter Ligation

On Tuesday, 11/12/19, we started our lab session. Our first task was to conduct double-digest restriction associated DNA sequencing. I labeled 2 tubes as P5 and P6, for my samples CHIM 002 and PRBM 005. Then, I added 6uL of each of my samples’ DNA in the corresponding tube. We prepared a master mix with 11 reactions using 9.9uL of CutSmart buffer 10X, 3.1 uL of EcoRI-HF enzyme, 1.3 uL of MSPI enzyme and 18.7 uL of pure water and we placed the master mix on ice. I added 3uL of master mix to each sample and sealed, vortexed, centrifuged and incubated the tubes at 37 degrees celsius for 8 hours. After that, we started our second task, adapter ligation for RADseq. I added 1uL of the working stock EcoRI adapters , EcoRI_8 and EcoRI_9, to my samples with sample IDs 15 and 16. We prepared an adapter ligation master mix with 11 reactions using 4.4 uL CutSmart buffer 10X, 14.3uL ATP(10mM), 2.2 uL T4 Ligase, 1.1 uL pure water. Then, I added 3uL of master mix to the digested DNA and sealed, vortexed, centrifuged and incubated the tubes at 16 degrees celsius for 6 hours.

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