Dr. Hiranya S. Roychowdhury
hroychow at nmsu.edu
Mon Oct 16 20:29:10 EST 2000
Rodney, assuming that I have understood your question correctly, this is it:
Usually the fill-ins are done with the Klenow (Large fragment of E.coli
DNA polymerase I). This works fine when a digestion generates 5' overhang.
Alternatively, T4 DNA polymerase is used and it can take care of both 5'
and 3' overhangs. The reaction is carried out usually in the same buffer as
was used for the digestion. The buffering conditions are:
5 to 50mM Tris.Cl (or Tris-acetate) pH7.5 to 8, salt (K+ or Na+) 50 to
100mM and Mg++ at 10mM to 50mM. These are the conditions that have worked
for me quite nicely. In other words, as long as you have your DNA in a
buffer containing at least 10mM Mg+ the fill in reaction works fine. The
most important constituent of the reaction is the dNTP mix which needs to
be in sufficient quantity; otherwise the exonuclease function of the DNA
polymerase will dominate. The rule of thumb is to include about 0.5mM of
each since rarely do the DNA end conc. exceed that. Most people use a 5 or
10mM dNTP mixture (if you have a tube with your PCR kit, use that).
However, if you know the composition of the overhang, you can simply
supplement with only those ntds. For example, the end generated by EcoRI
(AATT) will only require dATP and dTTP.
Here is the basic protocol:
DNA (in RE buffer) 20uL
10mM dNTP 1uL
Polymerase 1uL (~0.5U)
incuabte 15-20min at RT (for Klenow) or 30min at 37C (for T4 polymerase)
and heat kill at 75C for 10min.
Some do PCI extraction following the fill-in.
It is always good to purify the filled-in DNA by some method that will
eliminate the unused dNTP's. I use simple agarose gel for that.
If you have many further Q, feel free to ask. I will, though, point out
that the Maniatis (old and new) has this written up quite nicely :)
At 01:03 PM 10/16/00 +0100, Rodney Earl Pettway wrote:
>I would like to know how to do a fill-in reaction. I am a little "SLOW"
>so I am going to have to have it spelled out to me.
>have a great day!!!!!!!!
>Rodney Earl Pettway.
>University of Florida
>Institute for Food and Agricultural Sciences
>Department of Entomolgy and Nematology
>Surge Area Drive
>Gainesville, Florida 32611
Dr. Hiranya Sankar Roychowdhury
College Asst. Prof.
Dept. of Chemistry & Biochemistry
Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003
Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu
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