Concentrating Methylated DNA via Methyl Miner Kit for Samples ACR_13, ACR_209, ACR_778 and ACR_221

MBD Enrichment for Sheared Samples 13, 209, 778, and 221 Started this round of MBD Enrichment on 20190826 for the remaining 4 FACE_PUF ACR Sperm Samples.

First step was to write out the steps for the day so I would know how much 1X Bind/Wash Buffer to dilute, as it comes in 5X in the kit, that way it was ready when it was needed (3480µl needed, 3600µl was made to account for any error)

Preparing Beads

1. Pipetted up and down the Dynabeads M-280 Streptavidin to resuspend them
2. Made 4, 1.5mL tubes, each with 10µl of Dynabeads (the amount recommended for less than or equal to 1µg of DNA input)
3. Brought volume up to a total of 100µl with 90µl of 1X bind/wash buffer to each tube
4. Pipetted to mix
5. Placed tubes on long magnet rack and removed and discarded supernatant when clear
6. Removed tubes from rack and resuspended beads in 100µl 1X bind/wash buffer
7. Placed tubes on long magnet rack and removed and discarded supernatant when clear
8. Removed tubes from rack and resuspended beads in 100µl 1X bind/wash buffer

Coupling MBD-Biotin Protein to the Beads

1. Thawed MBD-Biotin protein from -80 on ice
2. Made 4 new 1.5mL tubes, each with 7µl of MBD-Biotin protein (amount recommended for 1µg of DNA input)
3. Added 93µl of 1X bind/wash buffer to each tube to get up to a total of 100µl
4. Transferred diluted protein to the washed bead tubes for a total volume of 200µl in each of 3 tubes
5. Put samples on the rotisserie mixer for 1 hour at room temp

Washing MBD-Biotin-Coupled Beads

1. Spun down briefly tubes from above
2. Placed tubes on magnet rack for 1 minute
3. Removed supernatant when clear
4. Resuspended beads in 100µl 1X bind/wash buffer and pipetted to mix
5. Mixed beads on rotisserie mixer for 5 minutes at room temp
6. Repeated: Spun down tubes briefly
7. Placed tubes on magnet rack for 1 minute
8. Removed supernatant when clear
9. Resuspended beads in 100µl 1X bind/wash buffer and pipetted to mix
10. Mixed beads on rotisserie mixer for 5 minutes at room temp
11. Repeated again: Spun down tubes briefly
12. Placed tubes on magnet rack for 1 minute
13. Removed supernatant when clear
14. Resuspended beads in 100µl 1X bind/wash buffer and pipetted to mix
15. Mixed beads on rotisserie mixer for 5 minutes at room temp
16. Finally: Spun down tubes briefly
17. Placed tubes on magnet rack for 1 minute
18. Removed supernatant when clear
19. Resuspended beads in 100µl 1X bind/wash buffer and pipetted to mix

DNA Capture Reaction

Steps 33-35 See Table |Sample|vol 5x Buffer (µl)|vol DNA (µl)| vol H2O (µl)| |—-|—–|—-|—-| |13|20|48|32| |209|20|48|32| |778|20|50|30| |221|20|42|38|

1. Transferred all of each diluted DNA sample to separate tubes with the MBD-Biotin bound beads for a total of 200µl in each tube and pipetted to mix
2. Mixed on rotisserie mixer overnight at 4 degrees C in the cold room (Started spinning at 11:51)

Aug 27th 2019, Removing Non-Captured DNA

1. Again started by writing out protocol to calculate how much 1X bind/wash buffer to dilute (800µl needed)
2. Took tubes out of the cold room rotisserie and spun down briefly
3. Placed tubes on magnet rack for 1 minute
4. Removed clear supernatant and SAVED in tubes labeled non-captured DNA and sample number
5. Resuspended beads in 200µl of 1X bind/wash buffer and placed on rotisserie mixer for 3 minutes
6. Spun down tubes briefly
7. Placed tubes on magnet rack for 1 minute
8. Removed clear supernatant and SAVED in tubes labeled wash and the sample number
9. Repeated: added 200µl 1X bind/wash buffer and placed on rotisserie mixer for 3 minutes
10. Spun down tubes briefly
11. Placed tubes on magnet rack for 1 minute
12. Removed clear supernatant and SAVED in the same tubes labeled wash and the sample number for a total of 400µl in each tube

Single Fraction Elution

1. Resuspended beads in 200µl High Salt Elution buffer
2. Incubated on rotisserie mixer for 3 minutes
3. Spun down tubes breifly and placed on magnet rack for 1 minute
4. Removed supernatant when clear and SAVED in a new 1.5mL tube labeled captured DNA and the sample number
5. Repeated: added 200µl High Salt Elution Buffer to resuspend beads in each tube
6. Incubated tubes on rotisserie mixer for 3 minutes
7. Spun down tubes briefly and placed on magnet rack for 1 minute
8. Removed clear supernatant and SAVED in each sample tube for captured DNA, for a total of 400µl in each tube

Ethanol Precipitation

Each tube for ethanol precipitation gets 1µl of glycogen (co-precipitator), 1/10th the volume of the sample of 3M sodium acetate pH 5.2, and 2 volumes of the sample 100% ethanol, so…

Sample	vol glycogen (µl)	vol sodium acetate (µl)	vol 100% EtOH (µl)
13 Non-Captured (NC)	1	20	400
209 Non-Captured (NC)	1	20	400
778 Non-Captured (NC)	1	20	400
221 Non-Captured (NC)	1	20	400
13 Washed Non-Captured (W)	1	40	800
209 Washed Non-Captured (W)	1	40	800
778 Washed Non-Captured (W)	1	40	800
221 Washed Non-Captured (W)	1	40	800
13 Captured (C)	1	40	800
209 Captured (C)	1	40	800
778 Captured (C)	1	40	800
221 Captured (C)	1	40	800

1. Vortexed to mix and spun down
2. Placed tubes in the -80 freezer for 2 hours
3. Set centrifuge to 1 degrees C with about 15 min time left and let it run to get down to temp First had to run centrifuge for about 5 minutes to get temperature down to 1 degree C before step 60 was completed
4. Prepared 4mL of 70% EtOH and put in -20 to chill down
5. Took sample tubes out of -80 and centrifuged for 15 minutes at 1 degrees C and 16,000 rcf
6. Took tubes out of centrifuge one at a time to keep cold, looked at tubes to see if there was a pellet. In all these steps tubes were taken out of the centrifuge one at a time to keep them cold
7. Removed the supernatant and discarded very carefully, and left a small amount of supernatant in the tube because pellet wasn’t always visibile and when it was it migrated towards the pipette.
8. Added 500µl of cold 70% EtOH to each tube carefully
9. Centrifuged for 5 minutes at 1 degrees C and 16,000 rcf
10. Very carefully removed supernatant from pellets
11. Centrifuged tubes for 5 minutes at 1 degrees C at 16,000 rcf
12. Removed any remaining supernatant as best as possible with the smallest pipette tip and making sure to not removed the pellet
13. Air dried pellet for 3 minutes
14. Resuspended pellets in 60µl of ultra pure water

High Sensitivity DNA Qubit

1. Followed Qubit protocol for HS reagents

Sample	Standard 1 (RFU)	Standard 2 (RFU)	1st reading (ng/ul)	Second reading (ng/ul)	Average ng/ul
13 C	43.28	26926.11	0.304	0.302	0.303
209 C	43.28	26926.11	0.198	0.196	0.197
778 C	43.28	26926.11	0.564	0.560	0.562
221 C	43.28	26926.11	0.510	0.506	0.508
13 NC	43.28	26926.11	6.04	6.02	6.03
209 NC	43.28	26926.11	1.01	1.05	1.03
778 NC	43.28	26926.11	5.80	5.78	5.79
221 NC	43.28	26926.11	5.84	5.82	5.83
13 W	43.28	26926.11	Too Low	Too Low	Too Low
209 W	43.28	26926.11	Too Low	Too Low	Too Low
778 W	43.28	26926.11	Too Low	Too Low	Too Low
221 W	43.28	26926.11	Too Low	Too Low	Too Low