Pico Methyl Seq Kit DNA Input Testings

Testing Zymo Pico Methyl Seq Kit Titrations from [Maggie’s Previous Post]

(https://meschedl.github.io/MESPutnam_Open_Lab_Notebook/PMS-Test/)

Today I used the sample ACR_221, aka Tube 9, from project FACE_PUF, which is an Acropora sperm sample from Mo’orea. This sample was sheared to the desired BP length of 643 from [Maggie’s Previous Post] (https://meschedl.github.io/MESPutnam_Open_Lab_Notebook/Sonication-Test/)

First I quantified the amount of DNA from this sample by Qubiting it

Sample Total DNA
Standard 1 179.27ug/mL
Standard 2 19856.03ug/mL
221 Cap 18.3ng/uL

Given the known DNA concentration of this sample, I then calculated the volumes of input DNA for the five input DNA concentrations of 100ng, 10ng, 1ng, 100pg, and 10pg All input DNA for this kit required a total volume of 20uL so

100ng of Input: 100ng/18.3 = 5.46 uL of DNA sample + 14.54uL of Ultra Pure Water

To keep all input volumes of sample ACR_221 consistent at 5.46uL I made four stocks of serial dilutions.

10ng dilution = 1uL of ACR_221 + 9uL of Ultra Pure Water ; vortex well and spin down 1ng dilution = 1 uL of 10ng dilution + 9uL of Ultra Pure Water ; vortex well and spin down 100pg dilution = 1 uL of 1ng dilution + 9uL of Ultra Pure Water ; vortex well and spin down 10pg dilution = 1 uL of 100pg dilution + 9uL of Ultra Pure Water ; vortex well and spin down

Sample Conc Volume of Input DNA uL Volume of Water Total volume for BS
100ng ACR_221 5.46uL 14.54uL 20uL
10ng ACR_221 5.46uL 14.54uL 20uL
1ng ACR_221 5.46uL 14.54uL 20uL
100pg ACR_221 5.46uL 14.54uL 20uL
10pg ACR_221 5.46uL 14.54uL 20uL

Section 1: BS Conversion Conversion Reaction

  • 20µl of each tube was taken and pipetted into new PCR tubes, one for each sample
  • 130µl of Lightning Conversion Reagent was added to each tube, vortexed, then spun down
  • The 5 tubes were put into the thermocycler bisulfite conversion program (under mes user), which is 98 degrees C for 8 minutes, then 54 degrees C for 60 minutes, then 4 degree C hold

Conversion Cleanup

  • Added 600µl of M-Binding buffer (lightning kit) to the provided Zymo-Spin IC Columns (4 of them, one per sample)
  • Added the bisulfite treated DNA (150µl) to its respective column and inverted to mix
  • Centrifuged at 12,000 rcf for 30 seconds and discarded flowthrough
  • Added 100µl M-wash buffer to each column and centrifuged at 12,000 rcf for 30 seconds
  • Discarded flowthrough
  • Added 200µl L-Desulfonation buffer to each column and let them sit at room temp for 15 minutes
  • Began warming elution buffer to 56 degrees C
  • Centrifuged for 30 seconds at 12,000 rcf and discarded flowthrough
  • Added 200µl M-wash buffer, centrifuged for 30 seconds at 12,000 rcf and discarded flowthrough
  • Repeated above wash step one time
  • Transferred columns to 1.5mL tubes
  • Added 8µl warmed elution buffer to the columns and let them sit for 1 minute
  • Centrifuged for 30 seconds at 12,000 rcf

Section 2: Amplification with PrepAMP Primers

  • Made Priming master mix on ice:
    • 2µl 5X PrepAmp buffer * 4.2 = 8.4µl
    • 1µl PrepAmp Primers (40µM) * 4.2 = 4.2µl -For 1ng sample had to use 1uL of 20uM (0.5uL of 40uM primer + 0.5uL of Ultra Pure Water)
  • Made new PCR tubes with 3µl of PrepAmp MM and 7µl of bisulfite treated DNA (100ng, 10ng, 1ng, 100pg, 10pg)
  • Kept those on ice
  • Made PrepAmp Mix on ice:
    • 1µl 5X PrepAmp buffer * 5.2 = 5.2µl
    • 3.75µl PrepAmp PreMix * 5.2 = 19.5uL
    • 0.3µl PrepAmp polymerase * 5.2 = 1.56µl
  • Set thermocylcer program with lid temp restricted to 25 degrees C and place samples inside and run:
    • 98 for 2 minutes
    • 8 degrees for 1 minute
    • 8 degree hold
    • During hold vortex, spin tubes down, add 5.05µl PrepAmp Mix to each tube, vortex, spin down, and place back in thermocycler
    • 8 degrees for 4 minutes
    • 16 degrees for 1 minute with 3% ramp rate
    • 22 degrees for 1 minute with 3% ramp rate
    • 28 degrees for 1 minute with 3% ramp rate
    • 36 degrees for 1 minute with 3% ramp rate
    • 36.5 degrees for 1 minute with 3% ramp rate
    • 37 degrees for 8 minutes
    • repeat back from the first step one time through again
    • During hold, vortex, spin tubes down, add 0.3µl PrepAmp Polymerase to each tube, vortex, spin down, and place back into thermocycler

    Section 3: DNA Clean and Concentrator Columns (DCC)

  • Made a 1.5mL tube for each sample, added 7:1 ratio DNA binding buffer, so 109.2µl of DNA binding buffer and 15.6uL of Product
  • Put elution buffer in thermomixer 56 degrees
  • Added DNA sample (15.6µl) to the appropriate 1.5mL tube
  • Vortexed, spun down, and added to the column
  • Centrifuged 12,000 rcf 30 seconds, discarded flowthrough
  • Added 200µl M-wash buffer to each column
  • Centrifuged 12,000 rcf 30 seconds, discarded flowthrough
  • Added 200µl M-wash buffer to each column
  • Centrifuged 12,000 rcf 30 seconds, discarded flowthrough
  • Transferred columns to 1.5mL tubes
  • Added 12µl warmed elution buffer to each column directly
  • Incubated 1 minute
  • Centrifuged 12,000 rcf 30 seconds

    Section 4: Second Amplification (Adapater Addition)

  • Made 1st Amp master mix:
  • 12.5µl 2X Library Amp Mix * 5.2 = 65.0µl
  • 1µl Library Amp Primer(10µM) * 5.2 = 5.2µl
  • Added 13.5µl MM to new PCR tubes
  • Added 11.5µl of cleaned and concentrated DNA sample to the appropriate new PCR tube
  • Vortexed, spun down, and placed in thermocycler programs specific to how many cycles each sample required: -100ng Sample 6 cycles -10ng Sample 8 cycles -1ng Sample 8 cycles -100pg Sample 10 cycles -10pg Sample 10 cycles

Cleanup with DCC

  • Made a 1.5mL tube for each sample, added 7:1 ratio DNA binding buffer, so 175µl of DNA binding buffer
  • Put elution buffer in thermomixer 56 degrees
  • Added DNA sample (25µl) to the appropriate 1.5mL tube
  • Vortexed, spun down, and added to the column
  • Centrifuged 12,000 rcf 30 seconds, discarded flowthrough
  • Added 200µl M-wash buffer to each column
  • Centrifuged 12,000 rcf 30 seconds, discarded flowthrough
  • Added 200µl M-wash buffer to each column
  • Centrifuged 12,000 rcf 30 seconds, discarded flowthrough
  • Transferred columns to 1.5mL tubes
  • Added 12.5µl warmed elution buffer to each column directly
  • Incubated 1 minute
  • Centrifuged 12,000 rcf 30 seconds

Section 5: Amplification with Index Primers

  • Made new PCR tubes for each sample with 12.5µl Library Amp Mix
  • Added indexed primers: -100ng Sample got 0.5uL of Index A -10ng Sample got 0.5uL of Index B -1ng Sample got 0.5uL of Index C -100pg Sample got 0.5uL of Index D -10pg Samplegot 0.5uL of Index E
  • Added 12µl of sample to the appropriate tube (all of the flowthrough from above DCC) note, 1431 captured had more that 12µl in the tube, portentially wash buffer that did not full come out? All of that sample was added to the tube for amplification.
  • Vortexed, spun down, and placed in theremocycler program 2nd Pico Methyl Amp program

Cleanup with DCC

  • Made a 1.5mL tube for each sample, added 7:1 ratio DNA binding buffer, so 175µl of DNA binding buffer
  • Put elution buffer in thermomixer 56 degrees
  • Added DNA sample (25µl) to the appropriate 1.5mL tube
  • Vortexed, spun down, and added to the column
  • Centrifuged 12,000 rcf 30 seconds, discarded flowthrough
  • Added 200µl M-wash buffer to each column
  • Centrifuged 12,000 rcf 30 seconds, discarded flowthrough
  • Added 200µl M-wash buffer to each column
  • Centrifuged 12,000 rcf 30 seconds, discarded flowthrough
  • Transferred columns to 1.5mL tubes
  • Added 12µl warmed elution buffer to each column directly
  • Incubated 1 minute
  • Centrifuged 12,000 rcf 30 seconds

D5000 TapeStation 100ng

10ng

1ng

100pg

10pg

Written on August 15, 2019