Protocol test: Round 2

Here we go again!

Step 0: Prepare for extractions

• Preheat the thermomixer to 56ºC
• Clean scalpel in 10% bleach solution

Step 1: Cut a tissue sample from the parrafin block

• I used Graham’s scale, which was way more sensitive
• Tared the scale with a piece of weigh paper
• Used a scooping thing to cut and scoop out parrafin-embedded tissue
  • Today I carved out waaay less tissue than I did yesterday! I think I definitely had too much tissue yesterday, which could have caused the lower yield
• Set the tissue on the weigh paper
• Weighed 0.0201 g of tissue

1-histologyblock

Figure 1. Histology block with tissue taken from the sample.

Step 2: Cut the block into smaller pieces and place them into a 2 mL round-bottomed processing tube

• Following the protocol’s suggestions, I cut the block into much smaller pieces than I did yesterday

Step 3: Add 1 mL xylene to the sample. Vortex vigorously for 20 s, and incubate for 3 min on the benchtop

Step 4: Centrifuge at maximum speed for 3 minutes (but do not exceed 20,000 x g)

• Maximum speed on the centrifuge was 13,000 RPM x g

Step 5: Remove the supernatant by pipetting. Do not remove any of the pellet

Step 6: Add 1 mL of ethanol (96%-100%, purity grade p.a.) to the pellet, and mix by vortexing for 20 s.

Step 7-8: Repeat Steps 4-5

Step 9: Open the tube and incubate at room temperature for 10 minutes, or until all residual alcohol has evaporated

• I incubated the sample for 25 minutes, just like yesterday

Step 10: Resuspend the pellet in 180 µL Buffer TD1.

Step 11: Add one stainless steel bead to each 2 mL processing tube, and place the tubes in the TissueLyser Adapter Set 2 x 24.

• Added about 50 µL of glass beads (1 small spatula full) into the 2 mL sample tube

Step 12: Operate the TissueLyser for 20 s at 15 Hz

• Instead, I vortexed the sample and glass beads at maximum speed for 25 s

Step 13: Carefully pipet lysates into new 1.5 mL microcentrifuge-safelock tubes

• The protocol suggested that I vortex the lysates vigorously to improve yield, so I vortexed the lysates for 20 s

Step 14: Add 20 µL proteinkinase K, and mix by pulse-vortexing for 15s

Step 15: Incubate for 1 hour at 56ºC using a shaker-incubater at 1400 rpm.

• I used the Genome Sciences thermomixer, set to 56ºC and 1400 rpm, for one hour.
• After the incubation ended, I increased the temperature to 80ºC to prepare for Step 18

Step 16: Briefly centrifuge the microcentrifuge-safelock tube to remove drops from the inside of the lid

• I centrifuged the tube for 15 s at 13,000 RPM x g
• There was no gelatinous pellet after incubation and centrifuging, so Proteinkinase K digestion did not go horribly

Step 17: Add 4 µL RNase A (100 mg/mL), mix by vortexing, and incubate for 2 min at room temperature

• I added RNase A
• Vortexed for 20 s at maximum speed
• Incubated at room temperature for 2 minutes
• Incubated at room temperature an extra 4.25 min while I was waiting for the thermomixer to reach 80ºC

Step 18: Incubate for 60 min at 80ºC at 1400 rpm

Step 19: Repeat Step 16

Step 20: Add 200 µL Buffer TD2, and mix by pulse-vortexing for 15 s

Step 21: Add 200 µL ethanol (96-100%) to the sample, and mix thoroughly by vortexing

• I mixed for 35 s using a vortex at maximum speed

Step 22: Repeat Step 16

Step 23: Pipet the sample, including any precipitate that may have formed, into the PAXgene DNA spin column placed in a 2 mL processing tube, and centrifuge for 1 min at 6000 x g. Plae the spin column in a new 2 mL procesing tube, and discard the old processing tube containing flow-through.

Step 24: Pipet 500 µL Buffer TD3 into the PAXgene DNA spin column, and centrifuge for 1 min at 6,000 x g. Place the spin column in a new 2 mL processing tube, and discard the old processing tube containing flow-through.

Step 25: Pipet 500 µL Buffer TD4 into the PAXgene DNA spin column, and centrifuge for 1 min at 6,000 x g. Place the spin column in a new 2 mL processing tube, and discard the old processing tube containing flow-through.

Step 26: Centrifuge for 3 min at maximum speed (but do not exceed 20,000 x g) to dry the membrane completely.

Step 27: Discard the processing tube containing flow-through. Place PAXgene DNA spin column in a 1.5 mL microcentrifuge tube, and pipet 50-200 µL Buffer TD5 directly onto the PAXgene DNA spin column membrane. Centrifuge for 1 min at maximum speed (but do not exceed 20,000 x g) to elute the DNA.

• I pipetted 50 µL on the spin column membrane and let it incubate for 5 minutes, then centrifuged for one minute at 13,000 RPM x g

Step 28: Assess DNA yield with the Qubit

• Obtain dsDNA BR standards from fridge
• Prepare the master solution, using a 1:200 ratio of dsDNA BR buffer to dye. The master solution is used for the two standards and the samples
  • Each sample needs 200 µL of master solution. Since I only had three tubes (2 standards, 1 sample), I needed 600 µL of solution
  • I prepared 660 µL of solution, using 656.7 µL buffer and 3.3 µL dye. 660 µL solution * 0.5 / 100 = 3.3 µL dye
• Pipet 200 µL master solution into each Qubit assay tube
• Add 10 µL of the correct standard to the standard assay tube
• Add 5 µL of sample to the sample tube
• Vortex the tubes for 2-3 seconds
• Incubate tubes at room temperature for 2 minutes
• Use Quibit to quantify yield
  • It’s pretty easy to follow the instructions on the machine! You select your assay (dsDNA BR), read standards, then load in the samples

Figures 2-4. Qubit results.

Results

Somehow I ended up with a lower yield than last time…1.16 ng/µL. I need 6000 ng total per sample to do MBD-Seq, which means 130 ng/µL. I need 100x more DNA!

Going forward

I posted another comment in this issue asking for guidance. One possible solution could be to increase the time I vortex with glass beads. I’ll try Thursday?