Title:  Restriction Digestion of Lambda Phage DNA and Gel Separation

 

By Kasandra Rose                                                                                         Dec 12, 2006

Purpose:        

To learn about restriction digestion preparation of DNA, and use the resultant bands to analyze DNA.  Also, to gain experience running an electrophoresis gel.  The effect of three restriction enzymes, EcoRI, BamHI, and Hind III, on Lambda DNA will be examined and a restriction map will be created for future reference.

Introduction:

Restriction endonucleases (RE) are the immune system of bacteria.  They recognize and cleave invading DNA preventing infection from viruses.  Although there are several types of endonucleases, only type two are normally found to be useful for scientific applications.  Type II endonucleases cut in predictable and repeatable ways, have no DNA modification capabilities and require only MG⁺⁺ and do not require ATP (as the other types of restriction endonucleases do).

Each RE has a specific DNA recognition sequence, which is usually palandromic.  For instance, Hind III, from Haemophilus influenzae, recognizes the DNA sequence AAGCTT and always cuts between the two adenosines.  This leaves a staggered cut of five overhanging bases.  All identical DNA will be cut identically if the RE is allowed to cut to completion.  However, within a species or even in one individual, there are allelic variations.  If one of these variations either creates or destroys a cut site, the cut pattern of the RE will change.

The main way of examining the pattern of cutting that a particular RE produces on a purified sample of DNA is to run an electrophoresis gel on the digested material.  An electrophoresis gel separates molecules based on size using an electrical field to attract (or repel) the molecules.  Since DNA is negatively charged due to the large number of oxygen molecules attached to the phosphate background, it is attracted to the positively charged pole.  The gel itself acts as a sieve and allows smaller molecules to move faster through it towards the anode while larger particles move more slowly.  Thus, the DNA particles are separated by their sizes with the smallest approaching the anode first.  If the charge is removed from the gel after the molecules have had time to spread out but before the smallest molecules have moved off the gel, distinct patterns of DNA are produced and the MW of each fragment can be estimated with the use of Ethidium Bromide (EtBr), which intercalates into and accumulates in the DNA, and UV light, which excites the EtBr.   It should be noted that EtBr is a mutagen and suspected carcinogen and appropriate safety precautions should be followed in its handling and disposal.

Materials:

• 0.8% Agarose gel with 5 ml/50 ml of EtBr  (See Appendix A for preparation instructions)
• TBE Electrophoresis buffer
• Loading dye (bromphenyl blue)
• Distilled water
• Restriction Buffer
• Restriction Enzymes:
• Bam HI
• Hind III
• Eco RI

l       DNA

Methods:

1-12-06

Four 1.5 ml tubes were labeled B, E, H and X (for no enzyme).

Tube B:

• Added 4 ml of l DNA
• Added 1 ml of restriction buffer
• Added 1 ml of Bam HI
• Added  4 ml of sterile distilled H₂O

 

Tube E

• Added 4 ml of l DNA
• Added 1 ml of restriction buffer
• Added 1 ml of Eco RI
• Added  4 ml of sterile distilled H₂O

 

Tube H

• Added 4 ml of l DNA
• Added 1 ml of restriction buffer
• Added 1 ml of Hind III
• Added  4 ml of sterile distilled H₂O

 

Tube X

• Added 4 ml of l DNA
• Added 1 ml of restriction buffer
• Added  5 ml of sterile distilled H₂O

 

All the tubes were placed in a 37° water bath for 1 hr.

The digested DNA was frozen in preparation to run on the gel the subsequent lab.

1-17-06

• Prepared Ladder
• Added 1 ml of Hind III ladder
• Added  8 ml of sterile distilled H₂O

 

• Added 1 ml of loading dye to each of the 5 tubes.

 

• Ran agarose gel with the following parameters:

1. All material loaded into appropriate lanes.
2. Power 125 volts, 30 mins

 

Lane                Sample

1                                     Ladder

2                                     Radhika

3                                     Radhika

4                                     Radhika

5                                     Radhika

6                                     Empty

7                                     Kas Uncut

8                                     Kas EcoRI

9                                     Kas Bam HI

10                                  Kas Hind III

 

Results

 

Figure 1:  Photo of gel taken 1-17-06 from experiment 1-12-06.

Discussion

 

The lanes pertinent to this discussion are the ones labeled L, U, E, B and H. For purposes of discussion, when referring to the bands, band one will be the band at the top for each lane with subsequent bands going downward so that the largest numbered band is at the bottom.

Lane L, for Ladder, appeared to have far too much DNA and yet even so the 4^th band down is very faint.   The cut pattern of the DNA in Lane H, for Hind III, should match this provided l Hind III ladder. Essentially it does.  However the fourth band down, the same one that was very faint on the ladder, is so faint as to be practically invisible in Lane L.  The DNA in this lane is not overloaded unlike the other lanes, which is partly due to a small amount floating out during loading.  The locations of the bands expected, and seen, for l cut by Hind III are 23k bp, 9.4k bp, 6.5k bp, 4.4k bp, 2.0k bp, 2.3k bp, and 0.5k bp. There is also a 0.125k bp. but it is very hard to see at any time.  In this experiment, the 0.5k bp has run off the bottom of the screen as well and was too faint to be noticed.

The DNA in lane U, which stands for uncut, appeared as a large smear instead of a bar across the gel.  This was exactly what would be expected for uncut DNA.

What would be expected to be seen in Lane E, cut by Eco RI, are five bands, one of which is a doublet (5804 and 5643 bp sizes) .  The other four expected bands would be at 21k bp, 7.4k bp, 4.9k bp, and 3.5k bp.  All of the experimental bands appear to satisfactorily match up to what is expected.  There are five discernable bands of DNA.  The 3^rd and 4^th band are somewhat blurred together but under magnification it is clear they are two separate bands.  There are two additional, unexpected, faint bands seen below the darker bands that indicate the primary DNA we were targeting.

Lane B, the l DNA cut by Bam, also matches up to expectations if you look only at the darkest bands.  The expected bands are at 16.8k bp, 7.2k bp, and two doublets of 6.5k/6.8k bp and 5.5k/5.6k bp.  There is an additional expected band formed by the sticky ends of the  l COS site which yields a fragment of 12.3k bp size.

Aside from the obvious overloading of DNA in most lanes, there appears to be some contamination of the DNA in lane E and lane B.  Further, the contaminating DNA may not be from the same source in both lanes.  The extra band in lane B between expected bands 2 and 3 made interpretation more challenging at first.  However, it does appear that the contamination in lane B, at least, matches contamination in the lane with DNA cut by Bam prepared by another person seen to the left.  (This would be the fourth band from the left which is unlabeled)  This may indicate contamination in the communal container of Bam restriction enzyme.  It cannot be from concatamers because no selection of bands would add up to around 9.4k bp where this stray band is located.