Has anyone had any luck trying to quantify Nextera XT libraries using qPCR? I have tried it twice with no success. I have been able to quantify TruSeq libraries and they have been working just right. It looks like primer sequences are correct, so I don't know what could be causing the problem.
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
Yes, we got a library, already normalized and eluted as single strands using the kit protocol. The protocol called for us to just heat denature it, snap cool and immediately load into the reagent cassette for sequencing. But we did a KAPA qPCR kit (not the Illumina-specific kit, just the generic one) using normal flow cell oligo primers. The result looked fairly reasonable, so we proceeded. Basically just did what the protocol asked. (Okay, I spiked in phiX out of paranoia -- but otherwise the followed the instructions on the box.)
Worked okay.
--
Phillip
-
You get better than 0.5x variation in read numbers using qPCR? Like you have several samples in a pool and the lowest one gave you 20 million and the highest gave you 30 million? That would be close enough to perfect to satisfy me!
I did not do the bead normalization myself -- we just got a Nextera XT pool from a customer and ran it. There were >20 different index pairs in it. I generally call it a "win" if the lowest sample gives > 1/2 the number of reads as the highest sample. So by that criteria, it was good. Actually, if I remember correctly, it was better than that.
--
Phillip
Comment
Latest Articles
Collapse
-
by seqadmin
Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...-
Channel: Articles
03-22-2024, 06:39 AM -
-
by seqadmin
The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.
Avian Conservation
Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...-
Channel: Articles
03-08-2024, 10:41 AM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 03-27-2024, 06:37 PM
|
0 responses
13 views
0 likes
|
Last Post
by seqadmin
03-27-2024, 06:37 PM
|
||
Started by seqadmin, 03-27-2024, 06:07 PM
|
0 responses
11 views
0 likes
|
Last Post
by seqadmin
03-27-2024, 06:07 PM
|
||
Started by seqadmin, 03-22-2024, 10:03 AM
|
0 responses
53 views
0 likes
|
Last Post
by seqadmin
03-22-2024, 10:03 AM
|
||
Started by seqadmin, 03-21-2024, 07:32 AM
|
0 responses
69 views
0 likes
|
Last Post
by seqadmin
03-21-2024, 07:32 AM
|
Comment