FAQ: Can I use this NEBNext kit with adaptors and primers from other vendors than NEB?

Yes, it is possible to use adaptors and primers from vendors other than NEB with this NEBNext kit. The adaptor must be a T overhang adaptor and the final library yield will be slightly lower than when using the NEBNext adaptors for Illumina®

Guidelines for use:

  1. Use 2.5 µl adaptor. Adaptors are typically used at 15-25 µM concentration. The optimal adaptor concentration and dilution would have to be determined experimentally.
  2. Skip the USER addition and the 15 min USER incubation. Add 3 µl water or TE to sample to make up volume difference for the next step. Note that when using the NEBNext Ultra (NEB #E7420) or Ultra II Directional RNA (NEB #E7760, #E7765) kits the USER step must be performed.
  3. Cleanup and size selection may have to be modified. If the current ratios in the manual are used, the resulting libraries will be approximately 50 bases shorter than stated in the table.
  4. PCR with NEBNext Q5®

Use Universal PCR Primers supplied with the adaptors used. Do NOT use NEBNext Index Oligo PCR primers. (5.0 µl each, 10 µM stock. Primer concentration may have to be optimized.)

Change the thermal cycling conditions: 

98°C for 30 sec - initial melting

STEP TEMP TIME
Initial Denaturation 98°C 30 seconds
X cycles of 98°C 10 seconds
60°C 30 seconds
65°C 45 seconds
Final Extension 65°C 5 minutes
Hold 4°C