Hi all,
We are doing the metagenomics study by using RT-PCR products as the samples for the libraries preparation. We are using NEBNext Quick DNA Sample Prep kit for the job.
We have selected the DNA fragments from 300bp to 800bp by gel method first, then we proceeded the library preparation as referred to NEB instruction manual. We have used Roche MID adaptors for the ligation step. We have done the qPCR (KAPA) to quantify the amount of molecules with adaptors properly ligated. Unfortunately, we found the yield (the amount of molecules with ligated adaptors) was poor which were not enough for downstream LV emPCR for FLX. Compared with sample from sheared genomic DNA for library preparation, the amount of the molecules from RT-PCR products were lower than in 10 to 100 fold.
We wonder anyone have similar experience and how to get through the problem. Did you think the method to prepare RT-PCR product can affect the ligation efficiency?
Thanks for your input
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
Latest Articles
Collapse
-
by seqadmin
The complexity of cancer is clearly demonstrated in the diverse ecosystem of the tumor microenvironment (TME). The TME is made up of numerous cell types and its development begins with the changes that happen during oncogenesis. “Genomic mutations, copy number changes, epigenetic alterations, and alternative gene expression occur to varying degrees within the affected tumor cells,” explained Andrea O’Hara, Ph.D., Strategic Technical Specialist at Azenta. “As...-
Channel: Articles
07-08-2024, 03:19 PM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 07-25-2024, 06:46 AM
|
0 responses
9 views
0 likes
|
Last Post
by seqadmin
07-25-2024, 06:46 AM
|
||
Started by seqadmin, 07-24-2024, 11:09 AM
|
0 responses
26 views
0 likes
|
Last Post
by seqadmin
07-24-2024, 11:09 AM
|
||
Started by seqadmin, 07-19-2024, 07:20 AM
|
0 responses
160 views
0 likes
|
Last Post
by seqadmin
07-19-2024, 07:20 AM
|
||
Started by seqadmin, 07-16-2024, 05:49 AM
|
0 responses
127 views
0 likes
|
Last Post
by seqadmin
07-16-2024, 05:49 AM
|