Ligation Efficiency test for Plate A, Pooling Pool 3 from Plate B

Date Performed: December 23rd, 2021

Ligation Efficiency Test Plate A

4 samples, made 5 for error
10ul Hotstart Ready Mix * 5 = 50ul
4ul nuclease free water * 5 = 20ul
.5ul 503 primer * 5 = 2.5ul
.5ul 703 primer * 5 = 2.5ul

• Pipette the ligation test sample(s) into new PCR strip tubes (40ul)
• Perform a 1.5X bead cleanup for each test sample. Briefly:
  • Add 60ul room temp KAPA Pure Beads to each sample and pipette mix 10X
  • Place on orbital shaker 15 min
  • Place on magnet rack and wait for solution to go clear
  • Remove clear solution without disturbing beads
  • Add 200ul fresh 80% ethanol to tubes
  • Remove solution from tubes
  • Add 200ul fresh 80% ethanol to tubes
  • Remove solution from tubes
  • Remove extra liquid with a p20 pipette
  • Wait ~2 minutes for excess ethanol to evaporate
  • Resuspend beads in 20ul Tris HCL
  • Place on orbital shaker for 5 minutes
  • Place on magnet rack and wait for solution to go clear
  • Remove clear solution from tubes into new strip tubes
• For each ligation test tube, there will be 2 PCR reactions, a 12 cycle and a 30 cycle PCR
• Make a PCR master mix on ice. Thaw ready mix and primers on ice. Vortex and spin down the ready mix and primers before use. Remember each ligation test gets 2 PCRs, so for 1 test samples the “n” number is 2.1 (error), for 2 test samples the “n” number is 4.2, etc. Use any primer set, 50X and 70X pair
  • 10ul KAPA HiFi HotStart Ready Mix * “n”
  • 4 ul nuclease free water * “n”
  • 0.5ul 50X primer * “n”
  • 0.5ul 70X primer * “n”
• Make one set of strip tubes for the 12 cycle PCR. Make a second set of strip tubes for 30 cycle PCR
• Into each tube in both strip tube sets that is going to be used (depends on how many test samples you have) add 15ul of the PCR master mix
• Add 5ul from the cleaned test sample(s) into both a tube in the 12 cycle set and the 30 cycle set
• Vortex and spin down the tubes meant for the PCR
• Use two thermocyclers, set the 12 cycle tubes in the thermocycler programed for the 12 cycle RAD LIG TEST program (in JONP Login). Set the 30 cycle tubes in the thermocycler programed for the 30 cycle RAD LIG TEST program
• While that is running, make a small 1% gel to set (gel protocol)
• After the program is done, run 10ul from each PCR reaction (use 2ul dye for 10ul sample) on the gel with a 1kb Plus DNA ladder for 1 hr at 80V

Plate B Pool 3

Full Instructions for 2 bead cleans are on previous post

When divided, each of the 4 1.5ul tubes had ~325ul (1.5X clean = 487.5ul beads) Qubit Results for First 1.5X Clean

Error when trying to run second qubit for first round of bead clean so remade standards

Qubit results from Second 1.5X Clean