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Old 02-14-2014, 01:40 AM   #1
Simone78
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Default adaptors and primers Nextera kit: modified or not?

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 Nexteraīs 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 doesnīt exist anymore. Is the PPC included in the PCR master mix? Or itīs not necessary, since the sequence of the 2 primers contained in the cocktail is the same as the 5ī-end of the i5 and i7 adaptors?
thanks for sharing your comments and opinions!
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Old 02-14-2014, 03:33 AM   #2
crocky
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Hi!
I do not have any experience with the Nextera kits, but I have been using the ChIP TruSeq kit. In this one the adaptors are modified for sure, and I think I managed to find the modifications anywhere. However, when I then wanted to order such modified oligos the price mounted up to only a bit less than the kit. Therefore I bought the kit, and I am very happy with it
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Old 11-18-2015, 08:30 PM   #3
devdoot
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Simone, I'm super grateful and using the homemade tn5 from your paper. However, using unmodified oligos, I'm having a similar problem to the one you describe above. Any advice?
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Old 11-18-2015, 11:22 PM   #4
Simone78
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Quote:
Originally Posted by devdoot View Post
Simone, I'm super grateful and using the homemade tn5 from your paper. However, using unmodified oligos, I'm having a similar problem to the one you describe above. Any advice?
Hi,
I am not 100% sure but I think that the Illumina adaptors have a biotin at the 5ī-end. We donīt buy our own adaptors anymore but just order those from Illumina. We never tagment more than 100-200 pg/sample and therefore donīt need to use them as they come but we rather dilute them 1:5. In this way they last quite long. Besides, the cost of these adaptors is extremely low compared to the cost of the Nextera kits.
/Simone
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Old 11-19-2015, 07:22 PM   #5
nucacidhunter
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Quote:
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 doesnīt exist anymore. Is the PPC included in the PCR master mix? Or itīs not necessary, since the sequence of the 2 primers contained in the cocktail is the same as the 5ī-end of the i5 and i7 adaptors?
The obvious difference is input amount and application. As you have noted PPC is pool of primers complementary to P5 and P7 flow cell binding region and is used in other Illumina kits such as DNA and RNAseq as well. It is necessary for Nextera DNA because the input is 50x more than XT and concentration of N5/S5 and N7 is not enough for optimum amplification of 50 ng input. PCR master mix for both kits is identical.
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Old 07-28-2016, 12:59 AM   #6
jingler
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Quote:
Originally Posted by nucacidhunter View Post
The obvious difference is input amount and application. As you have noted PPC is pool of primers complementary to P5 and P7 flow cell binding region and is used in other Illumina kits such as DNA and RNAseq as well. It is necessary for Nextera DNA because the input is 50x more than XT and concentration of N5/S5 and N7 is not enough for optimum amplification of 50 ng input. PCR master mix for both kits is identical.
Instead XT, we have Nextera DNA kit in our lab, however, we have very small amount of start material (For RNAseq). Any idea I could use Nextera DNA staff in our smartseq2 experiment?

Thanks

Last edited by jingler; 07-28-2016 at 01:42 AM.
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Old 07-28-2016, 04:10 AM   #7
nucacidhunter
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SMARTseq yield will be too low for standard or scaled down Nextera.
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Old 07-29-2016, 12:03 AM   #8
Simone78
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Quote:
Originally Posted by nucacidhunter View Post
SMARTseq yield will be too low for standard or scaled down Nextera.
yes, unfortunately itīs like that. Tagmentation <1 ng DNA input doesnīt work very well with the Nextera kit.
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