3 & 5' Overhangs...
andrew at qimr.edu.au
Fri Jun 3 00:31:19 EST 1994
David L. Haviland, Ph.D. (HAVILAND at KIDS.WUSTL.EDU) wrote:
: Hi fellow netters:
: -------------.. 3' overhang left by SacI
: and 5'
: ------.. 5' overhang left by NheI
: I'd like to be able to join these two ends and retain at the very least the
: SacI site. As I interpret the details of T4 Polymerase, T4 will only end
: fill the NheI site in the presence of dNTPs. WITHOUT dNTPs T4 will remove
: the 3' overhang of the SacI site.
And WITH the dNTP's present.
: Using T4 should, if I understand things correctly, give back the NheI site
: at the expense of the SacI site. Correct? Unless I'm missing something T4
: can *not* end-fill the SacI site? (It can't go 3'->5')
Yep, you'll get ---GCTAG and C---
....which restores the NheI site only.
: I'd like to be able to keep BOTH sites but now I'm beginning to wonder if
: that can even be done without the aid of a SacI-NheI linker (most likely
: home made!).
Whatever the reason you need Sac I there, you can't do it with end filling.
T4 RNA ligase might do it with the unfilled ends juxtaposing, then do a
nick repair with T4 DNA ligase (I haven't done it...just a suggestion) or
alternatively make a CTAGAGCT phosphorylated oligo to stick the ends together
email: andrew at qimr.edu.au
The Bancroft Centre, Queensland Institute of Medical Research
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