M2W FK Library prep, 6-14-17

KAPA Hyper Prep Kit – 250 ng per library (1 library)

1) End Repair and A-tailing

Qubit concentration of DNA pool: 23.7 ng/ul

Library:                          M2W-FK-6

ER and AT Buffer                 7 ul                                         *For 250 ng (250ng x 1ul/23.7ng = 10.5ul)

DNA (pool)*                         10.5 ul

ER and AT Enzyme              3 ul

H2O**                                  39.5 ul                                   **Tot vol must be 60ul (add H2O to get 60)

Total                                      60 ul

Incubate at 20C for 30 min, 65C for 30 min, 4C forever.

2) Adapter Ligation

NEXT FLEX ID:                      6 (CTTGTA)

H2O                                            5 ul

Ligation Buffer                         30 ul

Adapter (15 uM)*                     5 ul          *Adapter stock is 25 uM

DNA Ligase                               10 ul

ER and AT Product                   60 ul

Total                                        110 ul

* For 15uM adapter, solve for: (25uM)(x) = (15uM)(6ul) => 3.6 ul 25uM stock to 2.4 ul H2O = 6 ul 15uM stock

Incubate at 20C for 15 min.

3) Post Ligation Clean-up

M2W-FK-6

Adapter ligation product          110 ul

Ampure beads (0.8x)                 88 ul

Follow bead clean-up procedure. Elute in 35 ul.

Qubit: 5.74 ng/ul

4) Library Amplification

1x

2x HiFi                              25 ul

10x Primer                      5 ul

DNA Library                   20 ul

Total                               50 ul

 

Cycling parameters                                                             Cycles

Initial denature                            98C               45s                 1

Denature                                      98C               15s

Annealing                                     60C               30s                 2*

Extension                                     72C               30s

Final extension                           72C                1min              1

End                                                4C                 Forever

*Number of cycles chosen is based on amount of starting material (concentration of library pre-amp) and amount of final product needed. KAPA kit guidelines state that if starting with 100ng 3-4 cycles should produce 1ug. We started with ~115 ng (114.8 ng in 20ul – 5.74 ng/ul product). I chose 2 cycles (rather than 3-4) because we generally don’t need 1ug of amplified library, and 2 cycles has been sufficient in prior runs.

5) SPRI Clean-up

Library Amp Product             50 ul

Ampure beads (1x)                50 ul

Follow bead clean-up procedure. Elute in 35 ul.

Qubit: 10.7 ng/ul

M2W_FK_library

Above is the gel image of the library both before and after amplification.

The final library product was diluted to 10 nM (per sequencing facility instructions) and submitted for sequencing on 6-15-17.