Setting up the PCR reactions (NEB #E7140)
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This caution sign signifies a step in the protocol that has two paths leading to the same end point. |
1.1. Setting up the PCR reactions (24 Reaction kit, NEB #E7140S)
1.1.1. Determine the number of libraries that will be amplified and pooled for subsequent sequencing.
1.1.2. Ensure that a valid combination of index primers is chosen based on color balance guidelines in the NEB #E7140 manual.
1.1.3. Thaw the EM-seq index primer tubes for 10 minutes at room temperature.
1.1.4. Briefly centrifuge primers supplied in individual tubes to collect all of the primer at the bottom of each tube.
1.1.5. Remove the appropriate amount of primer from each single use tube using individual pipette tips for each primer. Each tube
contains enough primer mix for one reaction.
1.1.6. Proceed with the PCR reaction according to the Enzymatic Methy-seq manual (NEB #E7120).
1.2 Setting up the PCR reactions (96 Reaction kit, NEB #E7140L)
1.2.1. Determine the number of libraries that will be amplified and pooled for subsequent sequencing.
1.2.2. Ensure that a valid combination of index primers is chosen based on color balance guidelines in the NEB #E7140 manual.
1.2.3. Thaw the NEBNext 96 Unique Dual Index Primer Pairs Plate for 10 minutes at room temperature.
1.2.4. Remove the hard-plastic plate cover from the 96 well plate. Briefly centrifuge the plate (280 x g for ~1 min) to collect all of the
primer at the bottom of each well.
1.2.5. Orient the NEBNext Unique Dual Index primer plate as indicated in Figure 1.1 (red stripe towards the user). With a pipette tip,
pierce the desired well(s) (Figure 1.1A) and transfer the volume of primer mix required for the PCR reaction to the PCR
plate/tubes. It is important to change pipette tips before piercing a new well to avoid cross contamination of indexed primers.
Alternatively, the wells can be pierced using the bottom of clean PCR strip tubes (see Figure 1.1B) prior to pipetting the primer
mix. Use a new, clean strip tube for each new well to be pierced.
Note: Each well contains one NEBNext Unique Dual Index primer pair. There is enough primer in each well for one PCR
reaction. Do not reuse primer if the seal has been previously pierced to avoid contamination with other indexed primers.
1.2.6. Proceed with the PCR reaction according to the Enzymatic Methy-seq manual (NEB #E7120).
