Part 2: Custom QXT

FIRST, IF POSSIBLE: Normalize cDNA samples to 2x the input mass (for 1ng cDNA input will be 2ng/10.13µL, for the 5ng input will be 10ng/10.13µL). Run quants (picogreen) to assess concentration. If normalization was successful, add one volume of water to the samples to bring it back to 1x concentration and continue with 10.16µL.

A	B	C	D	E	F
1ng cDNA input:			5ng cDNA input:
❏ INPUT: 1ng cDNA in 10.16uL water			❏ INPUT: 5ng cDNA in 10.16uL water
❏ QXT DILUTION: 1:20 dilution of QXT enzyme:Storage Soln., at least 2uL of dilution/sample			❏ QXT DILUTION: 1:6 dilution of QXT enzyme:Storage Soln., at least 2uL of dilution/sample
❏ MASTER MIX:			❏ MASTER MIX:
Reagent	1 rxn (uL)	____rxn	Reagent	1 rxn (uL)	____rxn
cDNA	10.16	N/A	cDNA	10.16	N/A
5x TD Buffer	8.84		5x TD Buffer	8.84
QXTEnzyme	0.1		QXTEnzyme	0.33
StorageSoln	1.9		StorageSoln	1.67
Total:	21		Total:	21

Place samples in thermocycler on “DNA Fragmentation” program:

A	B	C
Step	Temp(C)	Time(hh:mm:ss)
1	55	00:10:00
2	4	00:01:00
3	4	Hold

Add 5µL 0.2%SDS and 24µL water/sample.

A	B	C
Reagent	1 rxn (uL)	____rxn (uL)
0.2%SDS	5
H2O	24
Totals:	29

Seal, vortex, incubate at Room temperature for 0h 1m 0s.

A	B
Single cleanup	Double SPRI cleanup (experimental)
❏ Vortex beads and incubate @ RT for 30minutes	❏ Vortex beads and equilibrate @ RT for 30minutes
❏ Add 49uL bead mix/sample (1.0x)	❏ 0.5x cleanup (>600bp fragment removal): Add 25uL beads to each 50uL sample, vortex, briefly spin
❏ Seal samples, vortex, briefly spin down	❏ Incubate samples 5 mins @RT
❏ Incubate samples @RT 5 minutes	❏ Add samples to magnet rack, allow solution to clear, save supernatant to new clean tube (optional: save beads)
❏ Put samples on magnet rack, allow to clear, discard supernatant	❏ 0.2x cleanup (removal of <150bp fragments, rounding out total cleanup to 0.7x): Add 10uL bead mix/sample, seal, mix, incubate for 5 mins @RT
❏ Wash 1 with 200uL 80% EtOH	❏ Put samples on magnet rack, allow to clear, discard supernatant
❏ Wash 2 with 200uL 80% EtOH	❏ Wash 1 with 200uL 80% EtOH
❏ Remove last of the EtOH, allow samples to air dry for at least 10 minutes.	❏ Wash 2 with 200uL 80% EtOH
❏ Elute with 24uL H2O, incubate for 2 mins @RT	❏ Remove the last Ethanol and allow samples to air dry for at least 10 minutes
	❏ Elute with 24uL H2O, incubate for 2 mins @RT

Add 24µL Kapa Hotstart Master Mix/sample.

Add 1µL i7 primer and 1µL i5 indexing primer, or 2µL from a multiplex plate.

Seal, vortex, briefly spin down samples, put in thermocycler on “Pre-Capture PCR” protocol.

A	B	C	D
Step	Temp(C)	Time(hh:mm:ss)
1	68	00:02:00
2	98	00:02:00
3	98	00:00:30
4	57	00:00:30
5	72	00:01:00
6	Return to step 3	13x for 1ng cDNA inputs	11x for 5ng cDNA inputs
7	72	00:05:00
8	4	hold

Avoid S511 QXT i5 primer

Make sure beads are equilibrated to Room temperature.

Add 32.5µL-35µL Ampure beads/sample (0.65x-0.7x beads:sample ratio), seal, mix, brief spin.

Incubate sample/bead mix @Room temperature for 0h 5m 0s.

Move to magnet stand, allow solution to clear, discard supernatant.

Wash 1 with 200µL of 80% EtOH.

Wash 2 with 200µL of 80% EtOH.

Elute in 20µL water. Add water to samples, take off magnet, seal, mix, spin, incubate @Room temperature for 0h 2m 0s.

Transfer samples back to magnet stand, allow to clear, save supernatant in separate, clean tubes.

REPEAT Ampure bead purification 2 for a total of 2x bead cleanups.

Evaluate on Tapestation.