FILL-IN REACTIONS

"Fill-in" reactions are used to conv ert the 3'-OH ends on DNA molecule that have 5' overhanging ends to blunt ends. Fill-in reactions are inefficient for DNA with 3'overhanging ends.

  1. Concentrate the DNA via ethanol precipitation & resuspend it in 8µl 1x H buffer.
  2. Add:
    2.0 µl total volume dNTPs (i.e. 0.5µl of each 20 mM stock)
    2.8 µl H    2O
    0.6 µl Klenow (0.9 U/&# 181;l)
    0.6 µl 10x H buffer
    14 µl
  3. Incubate RT, 25 minutes. Stop reaction with 1µl 0.2 M EDTA & 20 µl H2O.
  4. Phenol extract by adding 35µl phenol, vortex, let stand at RT 5 minutes. Re-vortex and spin in a microcentrifuge for 1 minute. Collect and extract the upper phase 3-4 times with ether. Ethanol precipitate the DNA. Centrifuge for 20 minutes at 4°C, wash the pellet once with 80% ethanol, centrifuge for 5 minutes and dry the pellet. Resuspend the DNA in 8 µl of TE.

RECIPES:
10x H buffer: (66 mM Tris-HCl pH 7.4; 66 mM MgCl2; 66 mM DTT; 0.5M NaCl)
132 µl 500 mM Tris-HCl stock, pH 7.4
66 µl 1M MgCl2 stock
66 µl 1M DTT stock
100 µl 5M NaCl stock
QS to 1 ml with H2O

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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Fill-In Reactions. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/fill-in.html). 1997. Oklahoma City. Revision Date: October 2, 1997."