Home Protocols Setting up the PCR Reaction (NEB #E6442)

Setting up the PCR Reaction (NEB #E6442)

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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.

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 (of the manual).

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 2 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 Primer Pairs plate.

 

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.

 

Index Pooling Guidelines

For a link to download a sample sheet with the index sequences for use with the Illumina Experiment Manager (IEM) please go to our FAQ's tab on www. neb.com/E6442 – NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (NEB #E6442).

For all HiSeq®/MiSeq® sequencers, Illumina uses a red laser/LED to sequence bases A and C and a green laser/LED to sequence bases G and T. For each cycle, both the red and the green channel need to be read to ensure proper image registration (i.e., A or C must be in each cycle, and G or T must be in each cycle). If this color balance is not maintained, sequencing the index read could fail. Table 2.1 lists some valid combinations (up to 8-plex) that can be sequenced together. For combinations > 8 choose any column and add any plex combinations as needed.

For the NovaSeq®/NextSeq®/MiniSeq® which utilize 2 color chemistry, valid index combinations must include some indices that do not start with GG in the first two cycles. Use Table 2.1 for some suggested combinations. Not all possible combinations are listed. Please confirm the color balance of the selected barcodes for low plex pooling. Please refer to table 2.2 for examples.Table 2.1

Table 2.2. lists each index sequence color coded to correspond to the red/green channel. For combinations of valid indices, ensure that you will have signal in both the red and green channels in each cycle. See below for examples of Good and Bad index combinations based on HiSeq/MiSeq guidelines:

 

Table 2.3. NovaSeq, NextSeq and MiniSeq use 2 color channel sequencing to simplify nucleotide detection. Clusters only in red or green are interpreted as C or T, respectively. Clusters in both red and green are read as A, while unlabeled clusters are G bases. For multiplexing a small number of samples, make sure the final index pool contains some indices that do not start with GG in the first two cycles. Listed here are some examples of good (signal in at least one channel for the first 2 cycles) and bad (the index read begins with GG) index combinations.

Table 2.4. lists each index sequence color coded to correspond to the red/green channel. For combinations of valid indices, ensure that you will have signal in both the red and green channels in each cycle.