Reaction Setup NEBNext® Q5U® Master Mix (NEB #M0597)

Reaction Setup:

  • Mix individual components prior to use.
  • Assemble all reaction components on ice.

COMPONENTS

50 µl RXN

FINAL CONC.

NEBNext Q5U Master Mix

25 µl

1X

Primers (Forward & Reverse)

5 μl (2.5 μl + 2.5 μl)*

10 μM each primer*

NEBNext EM-seq™

Adaptor-ligated, converted DNA

20 µl

Variable

* NEBNext Index Primer Pairs are supplied in #E7120 and #E7140.

  • Gently mix the reaction and collect the liquid at the bottom of the tube with a quick spin.
  • Transfer reaction to a preheated thermocycler (98°C).

Thermocycling Conditions:

STEP

TEMP

TIME

CYCLES

Initial Denaturation

98°C

30 seconds

1

Denaturation Annealing Extension

98°C

10 seconds

4–8*

62°C

30 seconds

65°C

60 seconds

Final Extension

65°C

5 minutes

1

Hold

4°C

&inbsp;

*The number of cycles depends on the input amount and library prep kit used.

Cycle Number Recommendations:

STARTING MATERIAL

INPUT AMOUNT*

CYCLES

Genomic DNA

200 ng

4

50 ng

5-6

10 ng

8

*Input amount is based on the amount of DNA used at the beginning of the EM-seq workflow, not the amount of DNA going into the PCR reaction.

Cycle number recommendations are based on optimization with the NEBNext Enzymatic Methyl-seq kit (NEB #E7120). Further optimization to avoid over amplification may be required.