Setting up the PCR Reactions (NEB #E6444)

Symbols

This caution sign signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the type of RNA input.

1.1. PCR Amplification

For < 96 samples, follow the protocol in Section 1.1A. For 96 samples, follow the protocol in Section 1.1B.

1.1A. Setting up the PCR reactions (< 96 samples)

1.1A.1. Determine the number of libraries that will be amplified and pooled for subsequent sequencing.

1.1A.2. Ensure that you choose a valid combination of barcode primers based on color balance guidelines in Section 2.

1.1A.3. Thaw the 96 Unique Dual Index Primers Plate for 10-15 minutes at room temperature.

1.1A.4. Remove the hard plastic plate cover. Mix briefly by vortexing and then centrifuge the plate (280 × g for ~1 min) to collect all of the primer at the bottom of each well.

1.1A.5. Orient the 96 Unique Dual Index Primers Plate Set 3 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 (see specific library construction manual for protocol). 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 a unique pair of index primers. 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.

1.1A.6. Proceed with the PCR reaction according to the specific library construction manual.

Figure 1.1. NEBNext Unique Dual Index Pairs Plate Set 3

1.1B Setting up the PCR reactions (96 samples)

1.1B.1. Thaw the 96 Unique Dual Index Primer Pairs plate for 10-15 minutes at room temperature.

1.1B.2. Remove the hard plastic plate cover. Mix briefly by vortexing and then centrifuge the plate (280 × g for ~1 min) to collect all of the primer at the bottom of each well.

1.1B.3. Orient the 96 Unique Dual Index Primer Pairs plate as indicated in Figure 1.1 (red stripe towards the user). With a pipette tip, pierce the wells (Figure 1.1A) and transfer the volume of primer mix required for the PCR reaction to the PCR plate (see specific library construction manual for protocol). 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 a unique pair of index primers. 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.

1.1B.4. Proceed with the PCR reaction according to the specific library construction manual.