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Second Test of Zymo Quick DNA Miniprep Extraction Kit on Horseshoe Crab Tissue

3 More DNA Extraction Tests on Horseshoe Crab Samples from Natalie, Trying Bead Homogenization

This time: Using Zymo bead lysis tubes and the tissuelyser instead of incubation for tissue breakdown. Also trying to cut smaller pieces of tissue Samples: 833, 819, 425 Extraction kit Beads

  • Thawed samples on ice
  • Made 3 bead tubes with 800ul of DNA/RNA Shield
  • 10% bleach, DI and ETOH forceps and scalpel
  • Placed new foil down for each tissue
  • Tried to cut smaller pieces of tissue and mince them well before putting the piece in the bead tube

833 1 425 2

  • Used new scalpel blade for each sample
  • Followed Tissuelyser suggestions for DNA extraction: 20 seconds at 15Hz
  • Samples were very bubbly afterwards, spun them down in the minifuge for a few seconds to collect liquid

833 3 425 4

  • The liquid was not very colored and samples weren’t looking very homogenized
  • Took out about 500ul of liquid and placed into new 1.5mL tubes
  • Because the tissue didn’t break up very well, I decided to try a second homogenization, and add the solid tissue buffer and pro K during homogenization
  • Added another 500ul of DNA/RNA shield to the bead tubes
  • Added 150ul solid tissue buffer and 10ul proK to the bead tubes
  • Homogenized again with same protocol

833 5 425 6

  • 500ul of 2nd homogenization liquid were removed form those bead tubes and placed into new 1.5mL tubes labeled with sample number and “2”
  • 1st homogenization tubes got “1” label and 250ul of solid tissue buffer and 15ul of Pro K. Vortexed and spun down
  • Proceeded with 6 extractions: set up 6 spin columns labeled with sample # and “1” or “2”
  • Added 1 volume of g-DNA binding buffer to each tube. For 1st homog tubes they got 750ul. For 2nd homog tubes they got 500ul
  • Vortexed and spun down all tubes
  • Added 700ul of each sample to their labeled spin columns
  • Centrifuged at 1300 rcf for 1 min
  • Discarded flow through
  • Added the rest of the liquid to the spin columns and centrifuged in the same way
  • Transferred columns to new collection tubes
  • Made 1.5mL tube with 10mM Tris HCl and warmed in thermomixer 55 degrees C
  • Added 400ul DNA pre-wash buffer to each column
  • Centrifuged 1300 rcf for 1 minute and discarded flow through
  • Added 700ul DNA wash buffer to each column
  • Centrifuged 1300 rcf for 1 minute and discarded flow through
  • Added 400ul DNA wash buffer to each column
  • Centrifuged 1300 rcf for 1 minute and discarded flow through
  • Made 1.5mL tubes labeled completely with sample names and information
  • Transferred spin columns to those 1.5mL tubes
  • Added 50ul of warmed 10mM tris HCl by dripping to each spin column filter
  • Incubated columns for 5 minutes
  • Centrifuged columns for 1 min at 1300 rcf
  • Repeated last 3 steps once
  • Placed tubes on ice afterwards

Qubit

  • Broad range dsDNA qubit (protocol)
  • Amounts are in ng/ul and samples were read twice
Sample Reading 1 Reading 2 Average DNA (ng/ul)
standard 1 198 - -
standard 2 24730 - -
819 1 27.6 27.2 27.4
833 1 33.8 34 33.9
425 1 5.9 5.8 5.85
819 2 12.3 12.2 12.25
833 2 12.7 12.8 12.75
425 2 2.78 2.72 2.75

1.5X bead clean of 1st extraction test

The smearing on the gel of the extraction of samples 388, 393, and 463 could be co-precipitated molecules and not DNA. A 1.5x cleanup should get rid of things that are not DNA.

  • Took out beads and equilibrated to room temp
  • Made fresh 80% EtoH
  • Thawed samples on ice
  • Added 150ul of KAPA beads to each tube and pipette mixed 10 times
  • Incubated on shaker 15 mins 200 rpm
  • Added tubes to magnet
  • Removed supernatent when clear
  • Added 500ul 80% ETOH
  • Removed supernatant
  • Added 500ul 80% ETOH
  • Removed supernatant
  • Let tubes dry for ~5 min
  • Took tubes off magnet and added 100ul 10mM Tris HCl to each tube and pipetted to mix
  • incubated on shaker 5 min
  • Placed back on magnet
  • Removed clear superntant into new tubes

Gel

  • Small 1% Agarose Gel (protocol)
  • Gel with all samples extracted so far

gel

Interesting that the bead clean up did not remedy the smearing, I guess that is just all DNA. The same smearing is in the other samples extracted today, the best one is 425, which had the smallest piece and no “exoskeleton” or other darker colored tissue piece. But that had the lowest concentration, not enough.

Written on July 30, 2020