Hi,
I am using Nextera XT kit from Illumina but tried to order the adaptors from another vendor, since the sequence is disclosed. Unfortunately, I get sub-optimal results due to accumulation of primer dimers after enrichment PCR.
I then ran a Bioanalyzer to compare the adaptors from the XT index kit with my (unmodified) adaptors and found out that the Nextera´s migrate as a fragment ca. 10 bp slower than the others. Obviously, they must be modified (blocked) somehow to prevent the formation of concatamers/dimers.
Furthermore, I was also wondering if anybody has ever noticed an interesting difference between the Nextera and the Nexetra XT kit. In the Nextera kit there was a tube of "PPC" (PCR Primer Cocktail) to add in the 4-primers enrichment PCR after tagmentation. In the Nextera XT kit this tube doesn´t exist anymore. Is the PPC included in the PCR master mix? Or it´s not necessary, since the sequence of the 2 primers contained in the cocktail is the same as the 5´-end of the i5 and i7 adaptors?
thanks for sharing your comments and opinions!
I am using Nextera XT kit from Illumina but tried to order the adaptors from another vendor, since the sequence is disclosed. Unfortunately, I get sub-optimal results due to accumulation of primer dimers after enrichment PCR.
I then ran a Bioanalyzer to compare the adaptors from the XT index kit with my (unmodified) adaptors and found out that the Nextera´s migrate as a fragment ca. 10 bp slower than the others. Obviously, they must be modified (blocked) somehow to prevent the formation of concatamers/dimers.
Furthermore, I was also wondering if anybody has ever noticed an interesting difference between the Nextera and the Nexetra XT kit. In the Nextera kit there was a tube of "PPC" (PCR Primer Cocktail) to add in the 4-primers enrichment PCR after tagmentation. In the Nextera XT kit this tube doesn´t exist anymore. Is the PPC included in the PCR master mix? Or it´s not necessary, since the sequence of the 2 primers contained in the cocktail is the same as the 5´-end of the i5 and i7 adaptors?
thanks for sharing your comments and opinions!
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