1X Bead Clean, Qubit, Digestion

Dates Performed: September 2nd, 9th, 14th

THIS FIRST SECTION IS JUST FOR THE FIRST ROW (A) OF PLATE B 1X Bead Clean

1. Thawed Plate B, vortexed, centrifuged
2. Set multi channel pipette to 51ul to remove Row A of plate B into set of strip tubes (10 samples)
3. Made fresh conical of 80% ethanol
4. Warmed 1000ul Tris HCL to 37C
5. Added 50ul beads to each sample with multichannel and mixed by pipetting
6. Put samples on orbital shaker for 15 minutes at 200RPM
7. Set up strip tubes for each waste removal (3 total)
8. Placed samples on magnet on shaker for 3 minutes
9. Once samples were clear, removed 90ul clear fluid and ejected into waste strip tube 1
10. Added 200ul 80% ethanol to each sample tube
11. Removed 200 ul ethanol from each sample tube and kept is strip tube 2 for waste
12. Added 200ul 80% ethanol to each sample tube
13. Removed 200 ul ethanol from each sample tube and kept is strip tube 3 for waste
14. Went back in with p20 set to 20 to remove any residual ethanol.
15. Let first 5 samples dry with caps off then added 70ul warmed Tris HCL to each sample
16. Opened 5 remaining tubes to dry then added 70ul warmed Tris HCL to each sample
17. Placed tubes back on shaker for five minutes
18. Placed tubes on magnet on shaker for two minutes until clear
19. Removed clear liquid and put in new set of strip tubes

Only made enough Qubit master mix for 8 samples

Sample	Run 1
S1	191.21
S2	22154.07
1	6.48, 6.76
2	6.60, 7.07
3	5.70, 5.90
4	5.88, 6.16
5	4.50, 4.70
6	2.52, 2.62
7	7.02, 5.76
8	5.62, 6.24

Made digestion master mix (enough for 12 samples) but still ended up needing to make more. I am unsure where this error is coming from, there should have been 20ul extra. Started digest in thermocyler 4 at 3:30

Post digest performed 1.5X bead clean

1. Removed strip tube from freezer to thaw and spin down
2. Made fresh conical of 80% ethanol
3. Added 120ul beads to each sample with multichannel and mixed by pipetting
4. Put samples on orbital shaker for 15 minutes at 200RPM
5. Placed samples on magnet on shaker for 3 minutes
6. Once samples were clear, removed 190ul clear fluid and ejected into waste
7. Added 200ul 80% ethanol to each sample tube
8. Removed 200 ul ethanol from each sample tube
9. Added 200ul 80% ethanol to each sample tube
10. Removed 200 ul ethanol from each sample tube
11. Went back in with p20 set to 20 to remove any residual ethanol.
12. Let samples dry
13. Added 33ul Tris HCL to each sample
14. Placed tubes back on shaker for five minutes
15. Placed tubes on magnet on shaker for two minutes until clear
16. Removed clear liquid and put in new set of strip tubes

Post 1.5X Clean Qubit Reads

Sample	Run 1
S1	181.22
S2	22197.32
1	9.20
2	8.90
3	7.34
4	7.92
5	6.08
6	3.40
7	7.26
8	7.22

Plate Reader Results from Row A

FOLLOWING SECTION FOR REMAINDER OF PLATE B

Followed 1X bead clean protocol. Did not warm Tris HCL and did not leave beads in. This plate had 7 almost full rows so I split the clean in half and did the entire clean on the top three rows then the entire clean on the bottom 4 rows. I think this should help with not having the beads dry too quickly. After adding 70ul Tris HCL I put the plate on the orbital shaker for ten minutes then on the magnet on the orbital shaker until the liquid was clear. Then I moved the clear liquid to a new plate for digestion. Since I have been running out of digestion master mix I made enough for 100 samples.

Number of columns	Volume to Tube
10	130
9	130
8	130
10	130
10	130
9	130
8	130