I am currently working on illumina sequencing sample preparation. For final PCR product, I check the same samples using invitrogen E-gel and bioanalyzer, but the DNA size from Bioanalyzer is always around 100bp larger than that on E-gel. I cut the 250bp band out of gel and qiagen column purified it, but I got a 350-400bp peak in Bioanalyzer. I am wondering if anybody here had the same experience and if this sample could still be sequenced. Thanks a lot.
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
thanks for the reply. Actually I run the E-gel DNA ladder and a regular PCR product (not an illumina sample) in the bioanalyzer. Both sizes are correct. I guess maybe my illumina samples contain some sort of complicated secondary structures. BTW, I forget to mention that the primers I used are not from illumina. I don't know if the illumina primers are modified so that they will give me the clean PCR product.
Comment
-
I got the same problem recently
Hi,
I am glad I am not the only one to get this. I also use none-illumina primer. At first, my libraries are fine and recently, the size shifted about 100bp up and I also suspect this is an artificial library. May I ask if what PCR reagent you are using? 2X phusion master mix or following the illumina protocol by adding enzyme, buffer, dNTP separately?
thanks d for the discussion.
Comment
-
I used phusion polymerase and added those reagents separately. Additionally, I don't think it's phusion's issue since I tried pfu as well and got similar products. I guess we got complicated secondary structures in our PCR product (not just linear double stranded DNA) which cause either gel or bioanalyzer size shift.
Comment
Latest Articles
Collapse
-
by seqadmin
The sequencing world is rapidly changing due to declining costs, enhanced accuracies, and the advent of newer, cutting-edge instruments. Equally important to these developments are improvements in sequencing analysis, a process that converts vast amounts of raw data into a comprehensible and meaningful form. This complex task requires expertise and the right analysis tools. In this article, we highlight the progress and innovation in sequencing analysis by reviewing several of the...-
Channel: Articles
05-06-2024, 07:48 AM -
-
by seqadmin
The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist...-
Channel: Articles
04-22-2024, 07:01 AM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, Yesterday, 06:57 AM
|
0 responses
11 views
0 likes
|
Last Post
by seqadmin
Yesterday, 06:57 AM
|
||
Started by seqadmin, 05-06-2024, 07:17 AM
|
0 responses
16 views
0 likes
|
Last Post
by seqadmin
05-06-2024, 07:17 AM
|
||
Started by seqadmin, 05-02-2024, 08:06 AM
|
0 responses
19 views
0 likes
|
Last Post
by seqadmin
05-02-2024, 08:06 AM
|
||
Started by seqadmin, 04-30-2024, 12:17 PM
|
0 responses
24 views
0 likes
|
Last Post
by seqadmin
04-30-2024, 12:17 PM
|
Comment