Ligation of short DNA fragments.
rfwhittier at hotmail.com
Mon Dec 16 21:43:30 EST 2002
I agree with Wo's suggestions, but there is one additional and important
caveat. Is your 2-base overhang palindromic, i.e. AT, TA, GC or CG? If so,
the short answer from Invitrogen's Technical Support is correct. As I
recall, SAGE does not use a palindromic overhang, and even if it did,
at least the ligated fragments themselves would differ, that is, you
would not be ligating the same fragment in a head to head or tail to
tail orientation. With your approach, you have a 50-50 chance with each
ligation of forming a large palindrome, which will form chi structures
that look like recombination intermediates and therefore be degraded
inside E. coli. Unless your overhangs force ligation as tandem direct
repeats, you are losing half your potential transformants with each
40mer extension of your repeat sequence. 0.5^12. You do the math.
>You also should use a ligation buffer that contains some viscuous stuff
>like PEG (e.g. the buffer from LifeTechnologies).
>>At 02:02 PM 2002/12/16 -0500, Zhongtang wrote: Hi every one,
>>I want to construc concatemers of 40-bp dsDNA fragments by T4 ligation.
>>Each of my short DNA fragments has a 2-base overhang on either end. I
>>tried twice, but I did not get enough ligation. Judged from gel
>>electrophoresis, my ligation products ranged from dimers (a lot) to about
>>400 bp (little). I need concatemers of >500 bp. The Technical Support with
>>Invitrogen told me that that will not happen. Does anyone out there have
>>similar experience, or suggestions? I got very large concatemers in SAGE
>>(with 4-base overhangs). Does the 2-base difference make such a difference
>>in ligation efficiency?
>>You suggestion and information will be very much appreciated.
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