Testing Digestion on 12 Samples

Date Performed: May 19th and 20th, 2021

Randomly chose 12 samples for digestion: 369,396,394,416,756,767,815,819,834,443-2,488-2,784,2 (Plate ZY) Started with Step 4 of ddRad Protocol (https://docs.google.com/document/d/1iWBGgBZuXlVuiEV-A3ecxeyn9ZaY389tAqGZWksjs5Y/edit)

1. Thawed samples, vortexed, and spun down
2. Added water to plate first then added corresponding amount of sample to get 350ng of DNA
3. Sealed plate, vortexed, spun down plate

Followed Bead Clean (1x) protocol

1. Made fresh conical of 80% ethanol (40mL 100% ethanol and 10mL of nuclease free water)
2. Added enough beads to trough. I used the old beads and the were definitely well mixed and room temperature
3. Added 50ul of beads to each sample and mixed by pipetting up and down until uniform
4. Put rubber cover on plate then placed on orbital mixer for 15 minutes
5. Put plate on magnet rack for 3 minutes until bead formed on side of tubes
6. Used the multichannel to remove 90ul of the clear supernatant from each tube
7. Added and removed 200ul 80% ethanol 2X
8. Let bead dry without becoming cracked
9. Added 70ul nuclease free water to each tube by dripping onto bead and mixing until resuspended and uniformally mixed.

Made master mix of digestion solution

• Vortex and spun down each component (made a lot extra for error)
• 200ul cutsmart buffer (8*25)
• 25ul PST1
• 25ul EcoR1

1. Vortexed and spun down master mix
2. Added 10ul of master mix to each sample
3. Pipetted to mix
4. Sealed plate
5. Started in thermocycler

May 20th - Bead clean and Qubit

1. Maggie took plate out of thermocycler and placed in 4 degree fridge
2. Arrived to lab and took out plate and Qubit standards.
3. Fixed PEG solution as described above (Maggie had taken this out for me at 3pm)
4. Made fresh 80% ethanol conical
5. Followed protocol for 1.5X bead clean:
   • Did not vortex and spin plate because the beads are still in
   • Added 120ul of PEG-NACL to each sample (solution was definitely room temp)
   • Pipetted up and down to mix until uniform.
   • Put rubber cover on plate then placed on orbital mixer for 15 minutes
   • Put plate on magnet rack for 5 minutes until bead formed on side of tubes
   • Removed 190ul clear supernatant from each well
   • Add 200ul 80% ethanol to each well
   • Remove 200ul of the supernatant from each well
   • Add 180ul 80% ethanol to each well
   • Remove 200ul of the supernatant from each well
   • Went back in with P20 to get any residual liquid
   • During all the above steps was very careful not to touch beads
   • After drying briefly, added 33ul of nuclease free water to each sample. This step always goes pretty poorly (see image below)
6. While plate was on orbital shaker I made the master mix for the Qubit
   • n = 12 + 2 + 2 = 16
   • 16 * 199 = 3184ul buffer
   • 16 ul Reagent
   • vortex and spun immediately after making and again before using
7. Placed plate back on magnet to easily remove 1ul for assay
8. Added 190ul master mix to each standard tube and 199ul master mix to each sample tube
9. Vortexed standards and added 10ul to corresponding axygen tubes
10. Added 1ul DNA to each corresponding axygen tube (for these I took them out and watched to make sure I could see the 1ul entering the liquid)
11. Vortexed and spun all standards and samples
12. Results from Qubit below:

MAGGIE GOT SAME RESULTS