adapter ligations (short oligos)...

rhubner at molbiol.ox.ac.uk rhubner at molbiol.ox.ac.uk
Tue Dec 19 16:26:19 EST 1995


Dear bionetters,

 anybody out there ever annealed short oligos (10-13 bp with one or two 4-base 
overhangs; hence, 6 or 9bp overlap "only") and purified them from ss 
molecules? 
 I managed to do this apparently with success for a 60bp stretch (had 55bp 
overlap)... nice band visible on PAA gel... however, for the smaller sized 
oligos I have troubles (no band visible, just background smear...) [BTW, 
background smear also present for 60bp DNA...] 
 The calculated Tm for the 9bp overlaps was 30oC and they are not palindromic 
over this sequence... so, RT manipulation should still be fine, right? (RT is 
about 23oC in lab... phew! ;-)...)
 I think ligating with the mix (unpurified) will scramble up the rx'n... 
alternatively, I might try ligating the ss-molecules with T4 RNA ligase and 
somehow anneal and hope for the bacteria to repair...

 Any advice/empirical info would be HIGHLY appreciated!
 Thank you sooo much   &  Merry Xmas to you all!

 Cheers,
 Roland




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