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Digestion and Cleanup Trobleshooting from ddRAD Protocol on Horseshoe Crab DNA

Testing Cleanup, Digestion, and 2nd Cleanup on Horseshoe Crab DNA with Water and 10mM Tris HCL

Natalie’s ddRAD library prep is coming out with no DNA after the cleanup after the restriction enzyme digestion. We have tried many things to figure out how the DNA is getting lost. In a previous test I had used 10mM Tris HCl that Amy had made a few months ago as the dilutant and the resuspention liquid on 4 samples. These resulted in DNA at the end! However, water should work in this protocol. I decided to try our currently open water (which we know doesn’t work but I was going to try one more time), open a new water, and make new 10mM Tris HCl out of both the new and opened waters. I don’t call the opened water “old” because this container was opened within the last 2 months.

Note: the previously opened water is Nuclease-free water. The newly opened water is ultrapure water. This distinction didn’t seem to matter. Both waters are certified DNase/RNase free.

1st Cleanup

  • New 50mL conicals were made with aliquots of opened and new water. 10mM Tris HCl was made with 50mL of opened or new water and 500ul of 1M Tris HCl.
  • A new plate was made with these components:
Sample Well ul of DNA (350ng) ul of Dilutant to 50ul Dilutant
851 A1 7.4 42.6 Opened H20
866 B1 4.1 45.9 Opened H20
862 C1 4.2 45.8 Opened H20
875 D1 16.9 33.1 Opened H20
881 E1 6 44 New H20
883 F1 17.5 32.5 New H20
443-2 G1 4 46 New H20
444-2 H1 6.4 43.6 New H20
446-2 A2 7 43 10mM Tris HCL made with opened H20
448-2 B2 4 46 10mM Tris HCL made with opened H20
796-2 C2 7.2 42.8 10mM Tris HCL made with opened H20
770-2 D2 6.2 43.8 10mM Tris HCL made with opened H20
780-2 E2 9.8 40.2 10mM Tris HCL made with new H20
859-2 F2 6.3 43.7 10mM Tris HCL made with new H20
870-2 G2 10.7 39.3 10mM Tris HCL made with new H20
836-2 H2 6.6 43.4 10mM Tris HCL made with new H20
  • A 1X bead clean was performed by adding 50ul of KAPA Pure Beads (old beads) to each well. The cleanup followed the basic bead clean protocol. The beads were re-suspended with 70ul of the same dilutant the sample got as above. The samples were not taken off the beads.

Digestion

  • Cutsmart buffer was thawed on ice, vortexed and spun down
  • PST1 and EcoRI were vortexed, spun down, and kept on ice
  • A digestion mix was made:
    • 8ul cutsmart * 17 = 136ul
    • 1ul PST1 * 17 = 17ul
    • 1ul EcoRI * 17 = 17ul
  • The master mix was vortex and spun down
  • 10ul of mix was added to each well in the plate
  • Each well was pipetted mixed with a p200
  • The plate was sealed with a foil seal and put in the thermocycler 12 hour digest program (37 degrees C for 12 hours then a 4 degree C hold)

2nd Cleanup Next Day

  • Used PEG NaCl made by Natalie on May 22nd
  • Added 120ul PEG to each well
  • The cleanup followed the basic bead clean protocol, except I used PEG instead of new beads because the beads were already in each sample
  • The beads were re-suspended in 33ul of the same dilutant the sample got in the original dilution
  • The 10mM Tris HCl samples re-suspended much better than the water ones:

Qubit

  • The plate was put on the magnet so that beads weren’t sucked up when preparing the Qubit tubes
  • Broad range dsDNA qubit (protocol)
Sample Reading 1 Reading 2 Average DNA (ng/ul) Dilutant
standard 1 171 - - -
standard 2 21506 - - -
851 Too low - - opened H20
866 Too low - - opened H20
862 Too low - - opened H20
875 Too low - - opened H20
881 Too low - - New H20
883 Too low - - New H20
443-2 Too low - - New H20
444-2 Too low - - New H20
446-2 13.3 13.2 13.25 10mM Tris HCL made with opened H20
448-2 9.52 9.46 9.49 10mM Tris HCL made with opened H20
796-2 11.4 11.6 11.5 10mM Tris HCL made with opened H20
770-2 10.4 10 10.2 10mM Tris HCL made with opened H20
780-2 9.66 9.66 9.66 10mM Tris HCL made with new H20
859-2 9.08 9.02 9.05 10mM Tris HCL made with new H20
870-2 11.6 11.6 11.6 10mM Tris HCL made with new H20
836-2 8.58 8.5 8.54 10mM Tris HCL made with new H20
Written on May 28, 2021