Endonuclease Digestion of DNA

Endonuclease Digestion of DNA

Endonucleases are the highly specific enzymes which recognize unique palindromic sequences in DNA at which they hydrolyze the phosphoester linkage. These enzymes have been crucial in the burgeoning fields of genetic sequencing and engineering. Because endonucleases are commonly found on the surface of the skin, as well in bacteria, latex gloves are routinely worn and good sterile technique is required when performing DNA manipulations.

We will perform three digestions on purified lambda phage DNA:
1) with Hind III (A|AGCTT), isolated from Hemophilus influenzae,
2) with Eco RI (G|AATTC), isolated from Escherichia coli, and
3) with both. [BamHI (G|GATCC) may also be used]

The resulting fragments can be used to construct a restriction map of the lambda genome.

All equipment and supplies listed in the protocol on Electrophoresis of DNA Fragments
37C incubator
Microcentrifuge tubes

Lambda DNA 0.5 ug/µL [50 ug]
(We have found it convenient to purchase lyophyllized
lambda DNA and rehydrate it when needed.
Eco RI endonuclease, 10 units/µL [2000 units]
Hind III endonuclease, 10 units/µL [2000 units]
specific buffers for Hind III and Eco RI
Hind III lambda digest, 0.125 ug/µL
loading dye

1. Prepare the digestion tubes in an Eppendorf microcentrifuge tube:

Single digestion:

0.0 µL ddH2O (enough to q.s. to 30 µL, depending on DNA conc.)
3.0 µL 10x buffer for the endonuclease you are using
17.0 µL Lambda DNA (at 0.286 ug/µL, = 5 ug DNA)
10.0 µL Eco RI or Hind III (10 units/µL = 100 units total enzyme)
total volume: 30.0 µL
Set up the double digestion:

0.0 µL ddH2O (enough to q.s. to 30 µL, depending on DNA conc.)
1.5 µL 10x buffer for Eco RI
1.5 µL 10x buffer for Hind III (It is probable that these two buffers are interchangeable.)
17.0 µL lambda DNA (at 0.286 ug/µL, = 5 ug DNA)
5.0 µL endonuclease (10 units/µL = 50 units of enzyme A)
5.0 µL endonuclease  (10 units/µL = 50 units of enzyme A)
total volume: 30.0 µL

2. Incubate at least 30 minutes at 37C, an hour might be OK, perhaps needed for double digestion.

3. Halt the reaction by placing on ice, add 6 µL of 6x loading dye, mix with same pipet, reset volume to 25 µL for next step (leave the tip in the Eppendorf tube used for digestion).

4. Load the wells: Pipet 5 µL into one well, reset pipet to 25 µL, pipet that volume into second well. On the same gel, run a lane with 1 ug undigested lambda DNA, and one with 5 ug of a Hind III digest as a standard.

Run the gel, photograph or carefully diagram the resulting bands.

Here are results of a 2006 experiment to test for optimum quantities of enzyme and optimum quantities of DNA.

Below is the gel which we produced Winter Quarter 2005.  Problems?  Of course…  lanes 3 and 12 (BamHI) should be identical.  12 appears to have been contaminated with another endonuclease.  Lane 4 shows some double digestion, but may not have been digested long enough.  Lane 7 and 13 (EcoRI) should be identical.  Lane 7 did not have sufficient endonuclease activity.   But, endonuclese activity was demonstrated, and evidence of double digestion developed  on all lanes with double enzymes (lanes 4, 9, and 14).

electrophoresis of double digestion, 2005

Click here to see a similar experiement from 2009.

Below are less successful results from 2003. Some browsers resize the image, so be certain that you are referring to the correct lane. Clearly there were several problems. We used two different batches of endonucleases which may explain why digestion was observed in some cases but not in others. The second from the top may be smeared because the last of the enzyme was rinsed out of the tube in order to get the last bit for the digestion.

2003 endonucleases results

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
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)

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