Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • JPC
    replied
    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?!

    Leave a comment:


  • yhey
    replied
    Originally posted by JPC View Post
    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
    I have heard recently from my FAS that one of the improvements to the enricher with the EZBead upgrade to v1.9, which has just been released, is to filter the E120 scale beads in several batches. Currently it filters in one batch and with the large scale emulsions there are just too many beads which puts excessive pressure on the filter causing it to be inefficient.
    If you feel your libraries are OK then maybe this is your problem and you are loosing 'good' beads.

    Yvonne

    Leave a comment:


  • JPC
    replied
    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:


  • pmiguel
    replied
    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:


  • RickC7
    replied
    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:


  • JPC
    started a topic E120 reactions

    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

  • seqadmin
    Latest Developments in Precision Medicine
    by seqadmin



    Technological advances have led to drastic improvements in the field of precision medicine, enabling more personalized approaches to treatment. This article explores four leading groups that are overcoming many of the challenges of genomic profiling and precision medicine through their innovative platforms and technologies.

    Somatic Genomics
    “We have such a tremendous amount of genetic diversity that exists within each of us, and not just between us as individuals,”...
    05-24-2024, 01:16 PM
  • seqadmin
    Recent Advances in Sequencing Analysis Tools
    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...
    05-06-2024, 07:48 AM

ad_right_rmr

Collapse

News

Collapse

Topics Statistics Last Post
Started by seqadmin, 05-24-2024, 07:15 AM
0 responses
195 views
0 likes
Last Post seqadmin  
Started by seqadmin, 05-23-2024, 10:28 AM
0 responses
218 views
0 likes
Last Post seqadmin  
Started by seqadmin, 05-23-2024, 07:35 AM
0 responses
221 views
0 likes
Last Post seqadmin  
Started by seqadmin, 05-22-2024, 02:06 PM
0 responses
12 views
0 likes
Last Post seqadmin  
Working...
X