Plate A 1X Bead Clean and Digestion

Date Performed: November 30th, 2021

Followed 1X Bead Clean Protocol

Make a fresh 50mL conical of 80% ethanol. Use 40mL 100% ethanol and 10mL of nuclease free water
The volume of KAPA pure beads needed for a 1X cleanup is 50ul. With a multichannel add 50ul of beads to each well in the plate with sample. You can pipette out the right amount + some error into a trough for the beads (ex for 96 samples that’s 4,800ul, which can be increased to 4,900ul for a little extra)
Add beads slowly to avoid bubbles and pipette up and down at least ten times to mix the beads with the DNA. It should be homogeneously brown when mixed Change tips between every use
After adding beads, put the plate on the orbital shaker (next to the Qubit) at 200rpm for 15 minutes
After 15 minutes, put the plate on the white magnet rack and return it to the shaker for another ~3 minutes
The solution should be clear in each well with a small brown pellet on the side with the magnet
Remove the plate from the shaker and bring it back to the bench
Use the multichannel to remove 90ul of the clear supernatant from each tube: Slide the pipette tips down the side of the tube opposite the magnet as to not disturb the beads. Try to keep heights of the tips even across the channels. Pipette up slowly to avoid bead suction. Discard of liquid in a trough/basin
Make a basin with 80% ethanol
Add 200ul 80% ethanol to each well in the plate, keeping it on the magnet. Pipette onto the side of the wells opposite of the beads to avoid disturbing them
Remove the supernatant wash (200ul) and discard into the waste basin. Avoid pipetting on the side with the beads
Add 200ul 80% ethanol to each well in the plate, keeping it on the magnet/Pipette onto the side of the wells opposite of the beads to avoid disturbing them
Remove all the supernatant wash (200ul) and discard into the waste basin. Avoid pipetting on the side with the beads
Make sure all liquid is out of each well by going back in with the multichannel for the p20 pipette set to 20ul and suck up any extra liquid at the bottom of each well
Let samples air dry ~2 minutes (ethanol should evaporate but the beads should not crack)
Make a basin with 10mM Tris HCl, enough for each sample (70ul each)
Add 70ul of 10mM Tris HCl, adding directly to the bead pellet this time by dripping slowly onto the beads. Pipette up and down by dripping onto the beads to resuspend the beads in every well. This may be hard with the multichannel, continue pipetting for them all.
Place plate on orbital shake for 5 minutes at 200rpm
Bring magnet back to shaker and place plate on magnet for 2 minutes
Remove clear liquid from plate and put into new plate
If you are going onto the next step in the same day, leave the plate out. If you are done for the day, cover the plate in a foil seal and store -20 freezer\

Followed Step 6 for Digestion of Plate A The next step is to digest the DNA with the two enzymes (PST and EcoR1)
If your plate was in the fridge, take it out and spin it down to collect all the liquid in the large centrifuge
Thaw cutsmart buffer on ice and take out the 2 enzymes you need and keep them on the ice as well (depending on how many you’re doing at once, you may need more than one tube of each of these)
Once the buffers are thawed, vortex and spin them down. Same with the enzymes
Make a master mix on ice of the digestion mix. Generate an n number which is the number of wells/samples + ~5% for pipette error. Ex: the plate above has 72 samples and 5 for error is 77
8μl Cutsmart Buffer (10X) * n = 1μl enzyme1 * n = 1μl enzyme2 * n = Add the larger volume first, then the smaller ones
Vortex and spin down the mix and keep on ice
Each well gets 10ul of master mix (total volume in each well is 80ul). You can use the multichannel by making strip tubes to take from. Example: the plate above has 8 rows and 10 columns (or less). Set up 8 strip tubes to pipette by column. In each tube add the amount of total master mix volume needed by that row (plus a little extra). For 10 columns, ~103ul is needed. For 9 columns, 92ul is needed, and for 8 columns, ~82ul is needed.
Use the multichannel to add 10ul of enzyme mix to each well. Change tips between each well. The final volume in each well should be 80ul
Use a p200 set to 30ul to pipette mix 10 times
Seal the plate with a labeled foil seal
Centrifuge the plate briefly in the large centrifuge to collect all liquid
Put the plate in the thermocycler you have signed up for. Make sure you have signed up for this thermocycler overnight because the program goes for 12 hours
Login to the “mes” user with the password “8888”, put the plate in the thermocycler and close the lid
Choose the program “12 hr digest” and press start. This incubates at 37 degrees C for 12 hours and then goes to 4 degrees
After the program is done, it goes to a 4 degree C hold. The next morning the plate can be put in the 4 degree fridge for storage\