Protocol for NEBNext Direct® Cancer HotSpot Panel (NEB #E7000)

A "printer friendly" version of this protocol can be downloaded from the Protocol & Manuals tab on the NEBNext Direct Cancer HotSpot Panel product page.

Symbols

  This caution sign signifies a step in the protocol that has multiple paths leading to the same end point but is dependent on a user variable, like the amount of input DNA.
   Stopping points in the protocol

Starting Material: 10 ng–1 µg DNA*

*For detection of somatic variants we strongly recommend starting with a minimum of 100 ng DNA input.

   If you are starting with cell free DNA, skip DNA Fragmentation and go directly to Hybridization in Section 1.2.2.

1.1. DNA Fragmentation with Covaris®

Shear input DNA in 50 μl (total volume) of 1X TE. Follow the Covaris recommendations for instrument and microtube setup using the 200 bp target size protocol for shearing in 50 μl.
1.2. Probe Hybridization

 
  Follow Section 1.2.1 if using FFPE DNA. If you are not using FFPE DNA, skip to Section 1.2.2 (Hybridization).


1.2.1. FFPE Phosphorylation
1.2.1.1. Make a FFPE Phosphorylation master mix by mixing the following components for the appropriate number of reactions:

REAGENT PER REACTION WITH 10% OVERAGE
 FFPE Phosphorylation Buffer
 5 µl
 5.5 µl
 FFPE Phosphorylation Enzyme
 1 µl
 1.1 µl
 Total  6 µl
 6.6 µl

1.2.1.2. Gently mix the master mix by pipetting up and down 10 times.

1.2.1.3. Add 42 µl of fragmented FFPE DNA from Section 1 to a PCR tube/well.

1.2.1.4. Add 6 µl of FFPE Phosphorylation master mix. Gently mix by pipetting up and down 5 times.

1.2.1.5. Incubate at 37°C for 15 minutes with the heated lid set to 45°C.

1.2.1.6. Briefly spin down the reaction to collect the sample at the bottom of the tube/well. Place the tubes on ice.

1.2.1.7. Proceed directly to Hybridization in Section 1.2.2.

1.2.2.    Hybridization
1.2.2.1. Make a Hybridization master mix by adding the following components for the appropriate number of reactions:

REAGENT PER REACTION WITH 10% OVERAGE
 Hybridization Buffer
 47 µl
 51.7 µl
 Hybridization Additive
 20 µl
 22 µl
 Cancer HotSpot Baits
 5 µl
 5.5 µl
 Total  72 µl
 79.2 µl

1.2.2.2. Gently mix the master mix by pipetting up and down 10 times.

1.2.2.3. Add 48 μl of fragmented DNA from Section 1 or 48 µl phosphorylated FFPE DNA from Section 2.1 to a PCR tube/well.

1.2.2.4. Add 72 μl of Hybridization master mix to each sample for a final volume of 120 μl. Mix by pipetting up and down 10 times. Seal the PCR plate or cap tubes securely to avoid evaporation.

1.2.2.5. Run the following program with the heated lid set to 105°C and place the samples in the thermocycler when the block temperature reaches 95°C:

10 min @ 95°C
90min @ 60°C
Hold @ 60°C

1.2.2.6. While the samples are incubating, prepare Streptavidin beads (see Streptavidin Bead Preparation in Section 1.3).

1.2.2.7. After the incubation at 60°C and when Section 1.3 (Streptavidin Bead Preparation) is complete, unseal the tubes/wells, leave the samples on the thermocycler at 60°C with the lid open and proceed to Bead Binding in Section 1.4.

1.3.    Streptavidin Bead Preparation

1.3.1. Warm Streptavidin beads to room temperature (~15 minutes).

1.3.2. Vortex the Streptavidin beads to resuspend.

1.3.3. For each reaction, 75 μl of beads are required (82.5 µl with 10% overage). In a 2 ml Eppendorf tube, add the appropriate volume of beads for the number of reactions performed.

Note: Use multiple 2 ml tubes if performing more than 12 reactions. It is not recommended to exceed 1 ml of beads per 2 ml Eppendorf tube.


1.3.4. Place the tube(s) on a magnet and wait for the solution to clear (~1 minute). Remove the supernatant, and then remove the tube(s) from the magnet.

1.3.5. Add 150 μl of Hybridization Wash (HW) per reaction (165 µl with 10% overage) to the beads and resuspend by vortexing or pipetting.

1.3.6. Place the tube(s) on a magnet and wait for the solution to clear (~1 minute). Remove the supernatant, and then remove the tube(s) from the magnet.

1.3.7. Repeat steps 1.3.5–1.3.6 twice for a total of 3 washes.

1.3.8. Resuspend the beads in 30 μl of Bead Prep Buffer per reaction (33 µl with 10% overage).

1.3.9. Keep the beads at room temperature until probe hybridization (Section 1.2.2) is completed.

Note: For Sections1. 4–1.10, the thermocycler lid should be open and unheated. The PCR tubes can remain uncapped for ease of mixing. However, if it is preferred, tubes can be capped, and then decapped prior to mixing.


1.4.    Bead Binding

1.4.1. Vortex the washed Streptavidin Beads (from Step 1.3.9) in Bead Prep Buffer to resuspend.

1.4.2. Add 30 μl of resuspended beads to each reaction (from Step 1.2.2.7) while the samples are on the thermocycler at 60°C, and then mix gently by pipetting up and down 10 times.

1.4.3. Change the thermocycler temperature to 48°C and incubate the reactions for 10 minutes. 

1.4.4. Remove the samples from the thermocycler and place on a magnet. Wait for the solution to clear (~ 15 seconds), remove the supernatant, and then remove the samples from the magnet.

1.4.5. Add 150 μl of HW to each sample. Gently mix by pipetting up and down 10 times. Place the samples on a thermocycler set at 62°C (lid open) and incubate for 5 minutes.

1.4.6. Remove the samples from the thermocycler and place on a magnet. Wait for the solution to clear (~ 15 seconds), remove the supernatant and then remove the samples from the magnet.

1.4.7. Repeat Steps 1.4.5–1.4.6 for a total of 2 washes at 62°C.

1.4.8.  Add 150 μl of Bead Wash Buffer 2 (BW2) to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature for up to 30 minutes and proceed directly to 3´ Blunting of DNA in Section 1.5.
 
  If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4°C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to 3´ Blunting of DNA in Section 1.5.

1.5.    3´ Blunting of DNA

1.5.1. While the beads are suspended in BW2 buffer, make a 3´ Blunting master mix by adding the following components in a sterile nuclease-free tube for the appropriate number of reactions:

REAGENT PER REACTION WITH 10% OVERAGE
 3´ Blunting Buffer
 97 µl
 106.7 µl
 3´ Blunting Enzyme Mix
 3 µl
 3.3 µl
 Total  100 µl
 110 µl

1.5.2. Gently mix by pipetting up and down 10 times.

1.5.3. Place the DNA bound beads on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.5.4. Add 100 μl of 3´ Blunting master mix (from Step 1.5.1.) to each sample. Gently mix by pipetting up and down 10 times. Incubate the samples at 37°C for 10 minutes on a thermocycler with the thermocycler lid open.

1.5.5. Proceed immediately with the Post-reaction Wash (Section 1.5.6).

1.5.6.    Post-reaction Wash


1.5.6.1. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.5.6.2. Add 150 μl of Bead Wash Buffer 1 (BW1) to each sample. Gently mix by pipetting up and down 10 times. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet.

1.5.6.3. Add 150 μl of BW2 to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature and proceed directly to dA-Tailing in Section 1.6.

  If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4°C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to dA-Tailing in Section 1.6.

1.6.    dA-Tailing

1.6.1. While the beads are suspended in BW2 buffer, make a dA-Tailing master mix by adding the following components in a sterile nuclease-free tube for the appropriate number of reactions:

REAGENT PER REACTION WITH 10% OVERAGE
 dA-Tailing Buffer
 97 µl
 106.7 µl
 dA-Tailing Enzyme
 3 µl
 3.3 µl
 Total  100 µl
 110 µl

1.6.2. Gently mix the master mix by pipetting up and down 10 times and set aside.

1.6.3. Place the DNA bound beads on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.6.4. Add 100 μl of dA-Tailing master mix (from Step1.6.1) to each sample. Gently mix by pipetting up and down 10 times. Incubate the reactions at 37°C for 10 minutes on a thermocycler with the thermocycler lid open. 

1.6.5. Proceed immediately with the Post-reaction Wash (Section 1.6.6).

1.6.6    Post-reaction Wash

1.6.6.1. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.6.6.2. Add 150 μl of BW1 to each reaction and then mix gently by pipetting up and down 10 times. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the reactions from the magnet.

1.6.6.3. Add 150 μl of BW2 to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature for up to 30 minutes and proceed directly to 3´ Adaptor Ligation in Section 1.7.

  If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4°C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to 3´ Adaptor Ligation in Section 1.7.

1.7.    3´ Adaptor Ligation

1.7.1. While the beads are suspended in BW2 buffer, make a 3´ Adaptor Ligation master mix by adding the following components in a sterile nuclease-free tube for the appropriate number of reactions:

REAGENT PER REACTION WITH 10% OVERAGE
 Adaptor Ligation Buffer
 80 µl
 88 µl
 3´ Adaptor
 10 µl
 11 µl
 Ligase  10 µl
 11 µl
 Total  100 µl
 110 µl

1.7.2. Gently mix by pipetting up and down 10 times and set aside.

1.7.3. Place the DNA bound beads on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.7.4. Add 100 μl of 3´ Adaptor Ligation master mix (from Step 1.7.1) to each sample. Gently mix by pipetting up and down 10 times.

1.7.5. Incubate the samples at 20°C for 15 minutes on a thermocycler with the thermocycler lid open.

1.7.6. Proceed immediately with the Post-ligation Wash (Section 1.7.7).

1.7.7.    Post-ligation Wash

Note: The following wash steps are different than the Post-reaction Washes.

1.7.7.1. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.7.7.2. Add 150 μl of BW1 to each sample. Gently mix by pipetting up and down 10 times. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.7.7.3. Repeat Step 1.7.7.2 for a total of 2 washes in BW1.

1.7.7.4. Add 150 μl of BW2 to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature for up to 30 minutes and proceed directly to 5´ Blunting of DNA in Section 1.8.
 
  If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4°C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to 5´ Blunting of DNA in Section 1.8.

1.8.    5´ Blunting of DNA

1.8.1. While the beads are suspended in BW2 buffer, make a 5´ Blunting master mix by adding the following components in a sterile nuclease-free tube for the appropriate number of reactions:

REAGENT PER REACTION WITH 10% OVERAGE
 5´ Blunting Buffer
 97 µl
 106.7 µl
 5´ Blunting Enzyme Mix
 3 µl
 3.3 µl
 Total  100 µl
 110 µl

1.8.2. Gently mix by pipetting up and down 10 times.

1.8.3. Place the DNA bound beads on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.8.4. Add 100 μl of 5´ Blunting master mix (from Step 1.8.1) to each sample. Gently mix by pipetting up and down 10 times.

1.8.5. Incubate the samples at 20°C for 10 minutes on a thermocycler with the thermocycler lid open.

1.8.6. Proceed immediately with the Post-reaction Wash (Section1.8.7).

1.8.7.    Post-reaction Wash


1.8.7.1. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.8.7.2. Add 150 μl of BW1 to each sample. Gently mix by pipetting up and down 10 times. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.8.7.3. Add 150 μl of BW2 to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature for up to 30 minutes and proceed directly to 5´ Adaptor Ligation in Section 1.9.

  If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4°C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to 5´ Adaptor Ligation in Section 1.9.

1.9.    5´ Adaptor Ligation

1.9.1. While the beads are suspended in BW2 buffer, make a 5´ Adaptor Ligation master mix by adding the following components in a sterile nuclease-free tube for the appropriate number of reactions:

REAGENT PER REACTION WITH 10% OVERAGE
 Adaptor Ligation Buffer
 80 µl
 88 µl
 5´ UMI Adaptor
 10 µl
 11 µl
 Ligase  10 µl
 11 µl
 Total  100 µl
 110 µl

1.9.2. Gently mix by pipetting up and down 10 times.

1.9.3. Place the DNA bound beads on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.9.4. Add 100 μl of 5´ Adaptor Ligation master mix (from Step 1.9.1) to each sample. Gently mix by pipetting up and down 10 times.

1.9.5. Incubate the samples at 20°C for 20 minutes on a thermocycler with the thermocycler lid open.

1.9.6. Proceed immediately with the Post-ligation Wash (Section 1.9.7).

1.9.7.    Post-ligation Wash

Note: The following wash steps are different than the Post-reaction washes.


1.9.7.1. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.9.7.2. Add 150 μl of BW1 to each sample. Gently mix by pipetting up and down 10 times. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.9.7.3. Repeat Step 1.9.7.2 for a total of 2 washes in BW1.

1.9.7.4. Add 150 μl of BW2 to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature and proceed directly to Adaptor Cleaving in Section 1.10.
 
  If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4°C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to Adaptor Cleaving in Section 1.10.

1.10.    Adaptor Cleaving

1.10.1. While the beads are suspended in BW2 buffer, make a Cleaving master mix by adding the following components in a sterile nuclease-free tube for the appropriate number of reactions:

REAGENT PER REACTION WITH 10% OVERAGE
 Cleaving Buffer
 95 µl
 104.5 µl
 Cleaving Enzyme Mix
 5 µl
 5.5 µl
 Total  100 µl
 110 µl

1.10.2. Gently mix by pipetting up and down 10 times.

1.10.3. Place the DNA bound beads on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.10.4. Add 100 μl of Cleaving master mix (from Step 1.10.1) to each sample. Gently mix by pipetting up and down 10 times.

1.10.5. Incubate the samples at 37°C for 15 minutes on a thermocycler with the thermocycler lid open.

1.10.6. Proceed immediately with the Post-reaction Wash (Section 1.10.7).

1.10.7.    Post-reaction Wash

1.10.7.1. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.10.7.2. Add 150 μl of BW1 to each sample. Gently mix by pipetting up and down 10 times. Place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet.

1.10.7.3. Add 150 μl of BW2 to each sample. Gently mix by pipetting up and down 10 times. Keep the samples at room temperature and proceed directly to Library Amplification in Section 1.11.
 
  If you choose to stop the protocol at this point, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant, and then remove the samples from the magnet. Resuspend the beads in 100 µl of 1X TE. DNA bound beads can be stored in 1X TE for up to 16 hours at 4°C. When ready to proceed with the protocol, place the samples on a magnet and wait for the solution to clear (~ 15 seconds). Remove the supernatant and then remove the samples from the magnet. Add 150 μl of BW2 to each reaction, mix gently by pipetting up and down 10 times, and then proceed directly to Library Amplification in Section 1.11.

1.11.    Library Amplification

Note: Refer to Chapter 2 if using the NEBNext Direct Index Primer Mix Plate provided in NEB #E7000X. Refer to Chapter 3 for guidelines on index pooling.

1.11.1. Place the reactions on a magnet, wait for the solution to clear (~ 15 seconds), remove the supernatant then remove the reactions from the magnet.

1.11.2. Add 45 μl of molecular grade water to each reaction. Mix gently by pipetting up and down 10 times to completely resuspend the beads.

1.11.3. Add the following components to a sterile strip tube/well in a PCR plate:

REAGENT PER REACTION
 Q5 Master Mix
 50 µl
 Index Primer Mix
 5 µl
 Resuspended beads (from Step 11.2)
 45 µl
 Total  100 µl

1.11.4. Gently mix by pipetting up and down 10 times. Seal the PCR plate or cap tubes.

1.11.5. Run the following program with the heated lid set 105°C and place the samples in the thermocycler when the block temperature reaches 98°C (It is critical to ensure that the block temperature has reached 98°C before placing samples in the thermocycler):
 
CYCLE STEP TEMP TIME CYCLES
Initial Denaturation
98°C 30 seconds
 1
Denaturation
Annealing
Extension
98°C
62°C
72°C
10 seconds
15 seconds
20 seconds
 20-25*
Final Extension
72°C 5 minutes
 1
Hold 4°C    

*Follow the PCR cycle number recommendations listed in Table 11.1.

Table 11.1: PCR Cycle Number Recommendations.
 
INPUT DNA RECOMMENDED NUMBER OF PCR CYCLES
 1,000 ng
 20
 500 ng
 21
 100 ng
 23
 10 ng
 25

1.11.6. Proceed to Purify Amplified Fragments in Section 1.12.

  PCR reactions with beads can be stored for up to 16 hours at 4°C.

1.12.    Purify and Size Select Amplified Fragments

1.12.1. If you detect significant evaporation from the PCR reaction, bring the volume up to 100 µl with molecular grade water.

1.12.2. Vortex the Sample Purification Beads to resuspend.

1.12.3. Add 85 μl of Sample Purification Beads to the PCR reactions. Mix well by pipetting up and down at least 10 times.

1.12.4. Incubate for 10 minutes uncapped at room temperature.

1.12.5. Place the tubes/PCR plate on a magnet. After the solution is clear (about 2 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets (Caution: do not discard beads).

1.12.6. Add 200 μl of freshly prepared (same day) 80% EtOH while the tubes/plate are on the magnet. Incubate at room temperature for 30 seconds and then carefully remove and discard the supernatant.

1.12.7. Repeat Step 1.12.6 once for a total of 2 washes in 80% EtOH, ensuring that all of the supernatant is removed from each reaction.

1.12.8. Incubate the samples, uncapped (or unsealed), at 37°C for 5 minutes on a thermocycler with the thermocycler lid open to dry the beads.

1.12.9. Remove the tubes/plate from the thermocycler and resuspend the dry beads in 102 μl of water. Incubate for 2 minutes at room temperature.

1.12.10 Place the tubes/plate on a magnet and allow the solution to clear (about 2 minutes).

1.12.11. Transfer 100 μl of the eluted library to fresh tubes/plate and add 85 μl of Sample Purification Beads. Mix well by pipetting up and down at least 10 times.

1.12.12. Incubate for 10 minutes at room temperature.

1.12.13. Place the tubes/plate on a magnet. After the solution is clear (about 2 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets (Caution: do not discard beads).

1.12.14. Add 200 μl of freshly prepared (same day) 80% EtOH while the tubes/plate are on the magnet. Incubate at room temperature for 30 seconds and then carefully remove and discard the supernatant.

1.12.15. Repeat Step 1.12.14 once for a total of 2 washes in 80% EtOH, ensuring that all of the supernatant is removed from each well.

1.12.16. Incubate the samples, uncapped (or unsealed), at 37°C for 2 minutes on a thermocycler with the thermocycler lid open to dry the beads.

1.12.17. Remove the tubes/plate from the thermocycler and resuspend the dry beads in 30 μl of 1X TE by gently pipetting (or gently vortex capped tubes/sealed plate, followed by a quick spin). Incubate for 2 minutes at room temperature.

1.12.18. Place the tubes on a magnet and allow the solution to clear (about 2 minutes).

1.12.19. Transfer 28 μl of the eluted library to a fresh tube.

1.12.20. Evaluate 1 μl of the eluted library on High Sensitivity Bioanalyzer Chip.
Figure 1.1: Examples of libraries prepared with human DNA (NA19240).