I'd like to order some custom oligos for multiplexing using the Illumina adapter/primer sequences, but am not sure what purification level to get (desalt or HPLC). Any experiences/recommendations with homemade primers? Do the deletion/truncation products left after desalting interfere with downstream reactions?
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
Late reply:
Illumina states "HPLC is what we use in the lab".
In the trenches, the guys in my core facility says desalted is fine.
Just make sure your Tm is ~74 degrees, and calculate using the Illumina primer as your comparison regardless of the program you use to calculate Tm.
Comment
-
Originally posted by SperSeq View PostHi,
I have the same question. Did you have any feedback? Maybe,since 2 years have passed, you are now the expert. Do you have any recommendation from your experience?
Thanks!
Comment
-
WE've been using standard desalted custom sequencing primers for over a year now, and have had excellent results...
Just remember to get the Tm up to ~70 degrees, so if this requires longer primers, choose a vendor that guarantees the quality of the synthesis...OR check by sequencing a PCR product, cloned into E.coli, derived from your Illumina-ready samples.
Z
Comment
-
When we build our own adapters, we perform this using standard desalted primers as well.
We add about half of the illumina adapter seq in a primary PCR (we're sequencing a PCR product whose 5' and 3' ends are known and constant), and then install the ends of the illumina adapters using a second PCR step.
All of these primers are regular desalted ~40mers.
Comment
Latest Articles
Collapse
-
by seqadmin
Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...-
Channel: Articles
04-04-2024, 04:25 PM -
-
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 -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 04-11-2024, 12:08 PM
|
0 responses
25 views
0 likes
|
Last Post
by seqadmin
04-11-2024, 12:08 PM
|
||
Started by seqadmin, 04-10-2024, 10:19 PM
|
0 responses
28 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 10:19 PM
|
||
Started by seqadmin, 04-10-2024, 09:21 AM
|
0 responses
24 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 09:21 AM
|
||
Started by seqadmin, 04-04-2024, 09:00 AM
|
0 responses
52 views
0 likes
|
Last Post
by seqadmin
04-04-2024, 09:00 AM
|
Comment