30. Title : Digestion and Ligation for sequencing#

30.1. Date#

21102020-

30.2. Objective#

To do all the steps in order to send my samples of WT, and dnrp1 strains to sequence after doing SATAY on them.

30.3. Method#

  • DNA concentration of 2ug for digestion

    • WT (~50ng/ul) = 40ul of DNA

    • \(\Delta\)nrp1_1 (~50ng/ul) = 40ul of DNA

    • \(\Delta\)nrp1_2 (~40ng/ul) = 50ul of DNA

  • Digestion samples:

    • 50ul final volume for WT and dnrp1_1

      • 40ul DNA

      • 5ul enzyme (250 units)

      • 5ul 10X buffer

    • 61uL final volume for dnrp1_2

      • 50ul DNA

      • 5ul enzyme (250 units)

      • 6.1ul 10X buffer

  • Ligation 1st step

    • 40uL 10X Buffer Ligase

    • 49uL digested sample

    • 5uL Ligase enzyme

    • 306uL MiliQ

  • Ligation 2nd step

    • 40ul 3M NaOAc

    • 1ul linear acrylamide

    • 1mL 100% EtOH

    • store in -20C

  • Resuspension of the DNA

    • Resuspend in 105uL MiliQ

    • Take 5uL to do a test 50uL PCR

    • Then add the PCR mix for 1mL to the 100uL

30.4. Results#

30.4.1. Digestion#

  • 1uL DNA loaded

30.4.2. PCR :)#

  • 50uL PCR

    • 5uL DNA

30.5. Conclusion#

  • The libraries are OK to send to sequence.