Another Overnight Gel and Zymoclean Large Fragment DNA Recovery Kit on 3mL-120hr HMW DNA Sample, Using Recommendations From Zymo Representatives

Notes

  • Using 3mL-120hr HMW DNA sample (~115ng/ul)
  • Skipped wells in the gel when loading to give ample room for slicing
  • Minimized time on UV,
  • I implemented all modifications to the Zymo kit to maximize DNA yield and length:
    • Heated elution buffer to 70 degrees C
    • Did two elution centrifuges
    • Incubated elution liquid on the column for >1 minute each time (5 min each)
  • Additionally I added a “dry spin” after the 2nd wash for 1 minute to dry the column of any residual wash buffer (ethanol)
  • From the Zymo Rep the suggested:
    • Incubating the gel slice while it dissolves for ~20min or until there were no swirls when tapping the tube
    • Increasing the centrifugation speed to 16,000rcf

20220103 Loading and Running Gel

  • 0.8% gel mix:
    • 180mL 1X TAE
    • 1.4g agarose
  • Microwaved for ~4min
  • Pipetted 1mL into a 1.5mL tube and saved on the heat block at 65 degrees C make sure the heat block is on and running
  • Made sure the large tray had well sealed tape barriers
  • Let the gel liquid cool for ~10 min until pouring into the tray and placing in the combs
  • Gel cooled in ~20 min or less
  • Added 1 round of PFG ladder to the left-most well
  • Let the 1mL saved gel cool for ~3 min outside of the heat block
  • Filled the PFG well with cooled gel liquid to the top of the well to seal the round in
  • Waited ~3 minutes for the wells to cool
  • Made up 48kb ladder:
    • 1.2ul ladder
    • 6ul loading dye
    • 28.8ul molecular grade water
  • Placed the gel tray into the box after removing the tape
  • Mixed 2ul of loading dye with 10ul of 3mL-120hr sample (with a clipped tip) and added it to the 3rd well in the gel
  • Added 16ul 48kb ladder to the 5th well in the gel
  • Set the timer for 16.5 hours at 40 volts and started it at ~5:02pm

20211208 Gel Slicing and Extraction

  • Stopped the gel at 9am
  • Sliced the gel in half and placed the half with samples in the EtBr bath for 1 hour
  • While that was in the bath, I made up final tubes and weighed tubes for the starting weight for the gel extraction
  • After 1 hour: put on PPE for looking at UV and took picture of the gel
  • Here I noticed the gel was a little strange (see image below), there is an upward tail of DNA that either is that large or ran funny that’s above the usual huge clump of HMW DNA. I decided to slice out that piece as a separate piece (“additional”), so I quick made up another tube for it and made measurements
  • Cleaned fresh razor blade with ethanol before use
  • Quickly sliced out a square of the whole “HMW” section of the 3mL-120hr sample, then made quick slices to cut the lower, middle, and upper pieces
  • Then slice out the top additional section
  • Turned off the UV for manipulating the pieces out of the gel and into their tubes
  • Gel image and approximate slice positions:
  • Looks like the gel ran kind of off angle again, because the 48.5kb positions in the ladders don’t line up, but if it did just run slightly off, then what I sliced should have the 150kb piece in it somewhere
  • Here is the gel without the slices in it:
  • Cleaned up and disposed properly all EtBr touching items
  • Weighted every sample tube with the gel in it, and calculated the gel weight, and the 3X gel weight
tube name tube weight (mg) tube weight with gel (mg) gel weight (mg) 3X gel weight volume (ul)
3mLC-150 984.1 1032.4 48.3 144.9
3mLC-UP 987 1046.4 59.4 178.2
3mLC-LOW 987.3 1013 25.7 77.1
3mLC-ADD 994.3 1077 82.7 248.1
  • Added 3 volumes of gel weight (5th column in table)of ADB to each sample tube (kit buffer)
  • Incubated sample tubes at 50 degrees C in the heat block for 20 minutes, then checked and most tubes still had a swirl in them, so they were let go for another 5 minutes. After this, all tubes but 3mLC-ADD were clearly dissolved. I kept that tube in the heat block until I had finished adding the other tubes to their spin columns
  • After this, the heat block was bumped to 70 degrees and elution buffer was incubated until later
  • Clipped tips of pipettes and added the total volume of each dissolved gel tube to individual spin columns and collection tubes provided by the kit
  • Centrifuged spin columns for 1 minute at 16,000rcf
  • Pipetted out flow through
  • Added 200ul of DNA wash buffer to each column
  • Centrifuged columns for 30 seconds at 16,000rcf
  • Pipetted out flow through
  • Added another 200ul DNA wash buffer to each column
  • Centrifuged for 30 seconds at 16,000rcf
  • Pipetted out flow through
  • Centrifuged for 30 seconds at 16,000rcf “dry”
  • Transferred columns to final 1.5mL tubes
  • Added 15ul of 70 degree C DNA elution buffer directly to the filters in the columns
  • Incubated the columns on the bench for 5 minutes
  • Centrifuged the columns for 30 seconds at 16,000rcf
  • Added 15ul of 70 degree C DNA elution buffer directly to the filters in the columns
  • Incubated the columns on the bench for 5 minutes
  • Centrifuged the columns for 30 seconds at 16,000rcf
  • Put the tubes on ice for Qubiting
  • Qubit HS DNA Assay:
    • 3mLC-150: 5.12ng/ul
    • 3mLC-UP: 7.42ng/ul
    • 3mLC-LOW: 2.14ng/ul
    • 3mLC-ADD: 8.13ng/ul