Bead Cleans and Digestions

Dates Performed: April 15th, 20th, 22nd 2021

April 15th, 2021: Bead Clean Plate C

• 1X bead clean (leave beads in!)

• volume=50ul

  Thawed plate C, vortexed and spun Made new 80% ethanol Followed 1X bead clean protocol (Step 5:https://docs.google.com/document/d/1iWBGgBZuXlVuiEV-A3ecxeyn9ZaY389tAqGZWksjs5Y/edit)

April 20th, 2021: Digestion Plate C and Bead Clean Plate B

Digestion Plate C:

• Thawed buffer, enzymes (PST1 and EcoR1), and plate c
• Vortexed and centrifuged

• Made master mix of buffer and enzymes Plate C has 37 samples – n= 37*1.05=39 Plate C master mix of enzymes o 8μl Cutsmart Buffer (10X) * n = 311 o 1μl Pst1 * n = 39 o 1μl EcoR1 * n = 39

  Strip tube setup for plate C for master mix

Number of columns	Volume to Tube
10	103
10	103
9	92
8	82

Followed Step 3 of above protocol.

Plate B Bead Clean (1X), followed above protocol, Maggie finished and put in fridge

April 22nd, 2021: Bead Clean Plate A and Digestions Plates A and B

Bead clean plate A, followed steps above

Digestions plates A and B Plates A and B have 74 – n = 74*1.05=78 (n = 156 for both plates)

Plates A and B master mix of enzymes o 8μl Cutsmart Buffer (10X) * n = 1248 o 1μl Pst1 * n = 156 o 1μl EcoR1 * n = 156

Strip tube setup for plates A and B master mix

Number of columns	Volume to Tube
10	103
10	103
9	92
8	82
8	82
9	92
10	103
10	103

Important Note:
For Digestion I did not make enough error margin, had to make more digestion mix for 4 samples
o 8μl Cutsmart Buffer (10X) * 4 = 32, 321.05 = 33.6
o 2μl Pst1 * 4 = 8, 81.05=8.4
o 2μl EcoR1 * 4 = 8, 8*1.05=8.4
Messed up multiplying the amount of digestion enzyme, I thought it was 2ul of each per sample which is how I got 8

Also Made PEG Solution on 4/22
*25mL 5mNACL
*10g PEG
*Up to 50ml Nuclease free water
Shaker to mix, foil, fridge