Athurva Gore/LabNotes/2011-4-8: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Ajgore
>Ajgore
Line 5: Line 5:
** DNA was definitely attached to beads, as normal amplification worked.
** DNA was definitely attached to beads, as normal amplification worked.
** DNA must have been detached
** DNA must have been detached
* '''Can we modify the experiment to still allow attachment to beads?'''
===Can we modify the experiment to still allow attachment to beads?===
** According to Invitrogen website [http://www.invitrogen.com/site/us/en/home/brands/Dynal/Streptavidin-Coupled-Dynabeads/dynabeads_streptavidin.html#How%20do%20I%20dissociate%20my%20biotinylated%20molecule], bead-detachment can occur when:
* According to Invitrogen website [http://www.invitrogen.com/site/us/en/home/brands/Dynal/Streptavidin-Coupled-Dynabeads/dynabeads_streptavidin.html#How%20do%20I%20dissociate%20my%20biotinylated%20molecule], bead-detachment can occur when:
*** Heat in formamide or basic solution at 95 for 2 min or 65 for 5 min
** Heat in formamide or basic solution at 95 for 2 min or 65 for 5 min
*** Boil beads
** Boil beads
* We can skip desulfonation step according to a published paper: [http://nar.oxfordjournals.org/content/35/1/e4.full?searchid=1&HITS=10&hits=10&resourcetype=HWCIT&maxtoshow=&RESULTFORMAT=&FIRSTINDEX=0&fulltext=tetzner]
** Basic solutions, formamide, EDTA, and heat in general are bad
** Can use 30 min at 95 C in initial reaction
** Specific detachment percentages: [http://www.invitrogen.com/site/us/en/home/brands/Dynal/Streptavidin-Coupled-Dynabeads/elution_of_the_streptavidin.html]
* '''What could be causing detachment?'''
** Bisulfite conversion involves adding an acidic solution with heat, incubation for a very long time
** Desulphonation buffer is a strong base; could be detaching DNA
** Other problems?
* '''Possible solutions:'''
** We can skip desulfonation step according to a published paper: [http://nar.oxfordjournals.org/content/35/1/e4.full?searchid=1&HITS=10&hits=10&resourcetype=HWCIT&maxtoshow=&RESULTFORMAT=&FIRSTINDEX=0&fulltext=tetzner]
*** Can use 30 min at 95 C as initial step in PCR; desulphonates DNA successfully without use of a base

Revision as of 22:42, 8 April 2011

Bisulfite Biotin-Binding

  • Ran gel for Bisulfite-converted bead-bound DNA and normal bead-bound DNA:

File:04072011 Low Input Bisulfite FAILED.jpg

  • Bisulfite-converted libraries did not work.
    • DNA was definitely attached to beads, as normal amplification worked.
    • DNA must have been detached

Can we modify the experiment to still allow attachment to beads?

  • According to Invitrogen website [1], bead-detachment can occur when:
    • Heat in formamide or basic solution at 95 for 2 min or 65 for 5 min
    • Boil beads
    • Basic solutions, formamide, EDTA, and heat in general are bad
    • Specific detachment percentages: [2]
  • What could be causing detachment?
    • Bisulfite conversion involves adding an acidic solution with heat, incubation for a very long time
    • Desulphonation buffer is a strong base; could be detaching DNA
    • Other problems?
  • Possible solutions:
    • We can skip desulfonation step according to a published paper: [3]
      • Can use 30 min at 95 C as initial step in PCR; desulphonates DNA successfully without use of a base