Athurva Gore/LabNotes/2011-4-8: Difference between revisions
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** 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?=== | |||
* 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 | |||
** 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 | ** 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
- We can skip desulfonation step according to a published paper: [3]