Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
We were told by AB to increase our titration point to 0.6picomoles this means our input library amount went from 3.62ng to 4.85ng for a 239 bp library. Unfortunately the Enricher (now running v1.9) shook the Hyb tube from it's place holder and the resulting Output tube solution is cloudy and not very brown....waiting to here from AB if I can collect the beads back together and save the emPCR somehow?!
-
Originally posted by JPC View PostHello All, is anyone running E120 reactions? I've tried 2 now and each time I only have 400M beads after enrichment instead of 2.2B.
We are using the EZ bead system and following the aqueous phase set up as per the calculator provided by AB (with a total of around 4.4ng of template, ave size 290bp).
What are the key factors in getting a successful reaction? if the instruments are set up correctly and the input material is correct what else can I tweak?
JPC
If you feel your libraries are OK then maybe this is your problem and you are loosing 'good' beads.
Yvonne
Leave a comment:
-
Thanks for the replies, we are quantifying with the Qubit, diluting our sample down to around 35pg/ul so that we are adding 125 ul of a 290bp product, I didn't want to dilute any further in case we drop below the qubit HS range.
'Yes' we are prep'ing in batches of 6 falcon tubes and 'No' we don't see nay bead fall out at all.
I'll give the qPCR a go to check the library, I have no reason to think there is a problem with it (we check regularly with the bioabnalyser, nanodrop and qubit) but it seems like a worthwhile exercise.
thanks both
JC
Leave a comment:
-
You probably should try qPCR on the library. EZ Bead tends to give some beads, even with no library (unlike the manual method). Your library may not be good.
--
Phillip
Leave a comment:
-
E120
Hi,
I have done several successful E120s, but when I have a problem it is usually too many beads...
How are you quantifying your library? If this is not accurate, then you will never get good beads.
Are you using a scale to prepare your oil mix or using the 6 falcon tubes? I heard some labs were having trouble with scales, so that's the reason for the falcon tube method.
Do you see any beads falling out of emulsion after amplification? Beads seen at bottom of pouch?
Just a few things I can think of...
Richard
Leave a comment:
-
E120 reactions
Hello All, is anyone running E120 reactions? I've tried 2 now and each time I only have 400M beads after enrichment instead of 2.2B.
We are using the EZ bead system and following the aqueous phase set up as per the calculator provided by AB (with a total of around 4.4ng of template, ave size 290bp).
What are the key factors in getting a successful reaction? if the instruments are set up correctly and the input material is correct what else can I tweak?
JPC
Latest Articles
Collapse
-
by seqadmin
The first FDA-approved CRISPR-based therapy marked the transition of therapeutic gene editing from a dream to reality1. CRISPR technologies have streamlined gene editing, and CRISPR screens have become an important approach for identifying genes involved in disease processes2. This technique introduces targeted mutations across numerous genes, enabling large-scale identification of gene functions, interactions, and pathways3. Identifying the full range...-
Channel: Articles
08-27-2024, 04:44 AM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, Yesterday, 08:02 AM
|
0 responses
10 views
0 likes
|
Last Post
by seqadmin
Yesterday, 08:02 AM
|
||
Started by seqadmin, 09-03-2024, 08:30 AM
|
0 responses
12 views
0 likes
|
Last Post
by seqadmin
09-03-2024, 08:30 AM
|
||
Started by seqadmin, 08-27-2024, 04:40 AM
|
0 responses
21 views
0 likes
|
Last Post
by seqadmin
08-27-2024, 04:40 AM
|
||
New Single-Molecule Sequencing Platform Introduces Advanced Features for High-Throughput Genomics
by seqadmin
Started by seqadmin, 08-22-2024, 05:00 AM
|
0 responses
358 views
0 likes
|
Last Post
by seqadmin
08-22-2024, 05:00 AM
|
Leave a comment: