ENDONUCLEASE DIGESTION OF DNA 

©David B. Fankhauser, Ph.D.
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
 Reagents for double
endonuclease digestion

This page has been accessed Counter times since 10 Feb 2004. 
16 March 1994, latest revision 27 Feb 1996, 3 Jan. '97, 18 Feb '97, 4 Jan '98, 16 Feb 01
electrophroesis of
double digestion (cosi, cosi)

This is the same protocol as the previous page from 2003, but performed with all fresh enzymes and freshly dissolved lambda DNA

Still room for improvement.
 

SAMPLE

10) undigested DNA

9) Hind III + Eco RI

8) Eco RI

7) Hind III

6) undigested DNA

5) Hind III + Eco RI

4) Eco RI

3) Hind III

2) undigested DNA

1) undigested  DNA

 VOLUME
in well
5 uL 

25 uL

25 uL

25 uL

25 uL

25 uL

25 uL

25 uL

25 uL

5 uL

 TEAM

B

B

B

B

B

A

A

A

A

A

 COMMENTS

Single, faint band

Bands appear different from those of 
Hind III, therefore some 2x digestions?
No bands apparent.  Either no DNA was added
or was completely digested by contaminants
Clear bands, altho lighter bands not apparent.
Perhaps too little DNA?
No single band.  Slight contamination from next
well
Massive overload of DNA.  Either because DNA
was not dissolved, or excess added to tube.
Single band seems to indicate lack of enzyme.

Bands typical of Hind III, correct amoung of
DNA present.
Correct amount of undigested DNA added for
clear single band.
Faint band present, 5 uL (of 1 ug DNA/30 uL)
not enough

 

 
 

There are eight Hind III fragments: 23,130; 9416; 6557; 4361; 2322; 2027; 564;125 bp long.
The 564 band is in the bromphenol blue, and the 125 band appears beyond the dye band.

There are five Eco RI fragments: 21,226; 7421; 5804; 5643; 4878; 5330

The following fragments were reported on the web as the double digestion products:
Double: 21,226; 5148; 4973;4268; 3530; 2027; 1904; 1584; 1375; 947; 831; 564

prep of reagents for electrophoresis
comb
eppendorf tube
DNA electrophoresis
Gel diagram
50x TAE buffer:
 

For 100 mL of 50x buffer:
 

24.2 g TRIS (base)
5.71 mL glacial acetic acid
10 mL 0.5 M EDTA (14.6 g/100 ml)