1. Restriction Enzyme Digestion of Phage DNA
Protocol 10.1 through 10.3
Objective: To cut phage genome into multiple fragments based on DNA sequence

3. General Introduction on Restriction Enzymes
Are also known as restriction endonucleases
Are naturally occurring enzymes used by bacteria for defensive purposes against extraneous DNA molecules
The enzymes cuts DNA at specific 4 to 6 base pair sequence, referred to as restriction sites that are palindromic
SEA-PHAGES currently uses the following:
BamHI
ClaI
EcoRI
HaeIII
HindIII
SalI

4. (OD260)(Dilution Factor) (50ng/µl)
DNA Quantification
Set Spectrophotometer to measure at 260nm and 280 nm wavelength
Blank instrument with water using glass cuvette only
Dilute a portion of extracted DNA 1:500 in sterile water with a total volume of 1ml
1/500 = X/1000µl
X=2µl of DNA 998µL water
Measure optical density reading of samples Calculate concentration of DNA by using the following formula:
(OD260)(Dilution Factor) (50ng/µl)

5. Determining DNA Concentration-
Based on the DNA concentration of your phage, you want to calculate the amount of DNA needed to set up the RE digestion
For example: if your DNA concentration is 125µg/ml, therefore to obtain ~ 0.5µg needed
Volume = DNA amount desired/concentration
0.5 µg(ml/125µg) =0.004ml = 4 µl
A Spectrophotomer- The Nanodrop 2000

6. Digestion Reaction Setup:
1. First, gently mix DNA tubes, then 65ᴼC for 10 minutes and then quickly place on ice. Gently mix DNA tubes, then 65ᴼC for 10 minutes and then quickly place on ice.
Enzyme 1
Enzyme 2
Enzyme 3
1. DNA (~0.5ug)
4uL
2. Buffer (10X)
2.5uL
3. Enzyme
0.5uL
4. Water
18uL
Total Volume
25uL
2. Set up master mix reaction tubes as shown in table, including negative control
Mix tubes gently and then spin down for less than 1 minute in a microcentrifuge
Then incubate 37ᴼC for 1 hour

7. Restriction Enzymes & Their Buffers
1.1
2.1
3.1
Cutsmart
BamHI +
ClaI
EcoRI
HaeIII
HindIII
SalI

8. General Overview
Relaxed phage DNA
65oC, ~5 mins

9. You can set up a master mix if you have many samples
Restriction Enzyme
Buffer
Sterile Water
Aliquot mixture into labelled microcentrifuge tubes and then add DNA last to prevent contamination
Mix contents gently and spin tubes quickly for less than 10 minutes, then incubate 37ᴼC for up to 1 hour

10. Gel Electrophoresis Materials needed Gel boxes and combs
Agarose (1 to 1.5%)
TBE buffer (1X)
DNA dye (e.g. Sybr Safe)
Loading dye
Erlenmeyer flask
Molecular weight marker

11. General Overview Load samples into wells with the loading dye
Remember to change tips between each sample
Cover, connect electrodes to power supply
Run until samples are out of the wells 80V for about 45 to 60 minutes

12. Fig. 10. 0-1. Restriction enzyme gel
Fig Restriction enzyme gel. An 8% agarose gel loaded with a 10kb ladder and phage DNA. Each lane labeled according to its content

13. Analyzing Restriction Enzyme Gels
Protocol 10.4
Objective: To examine and interpret result of restriction enzyme digest

14. DNA Ladder Fragment Size (bp)
Standard Curve
Table Sample data table for creating DNA standard curve for agarose gel shown in Figure 1 2 3 4 5 6 7 8 9
Distance Travelled
(mm)
DNA Ladder Fragment Size (bp) 15
10,000 17
6,000 20
3,000 23
2,000 27
1,500 31
1,000 50
500
Figure Sample restriction enzyme gel.

15. Determination of Band Size
Measure the distance from the well to each band in the ladder
Graph the distances vrs molecular weight on a semi- log graph with distance on the linear axis and molecular weight on the log axis
Create a line through each point to make a standard curve from which you will use to determine the molecular weight of the unknown bands on the gel

16. Example of Standard Curve to Calculate the molecular weight (MW) of Bands
Measure distance migrated of each band in marker.
Plot distance migrated vs. Log of #base pairs
Get equation of line.
Plug in distance of phage bands and calculate MW
Y=mX=B

17. Recognize the pattern the bands make and correlate to the sizes in the photo in the lab manual p.92
6000 bp
5000 bp
4000 bp
3000 bp
2000 bp
1650 bp
1000 bp
850 bp
650 bp
500 bp
400 bp
300 bp

19. Measure the distance from the well to each band in the lane and interpolate the size from the standard curve

20. A band which traveled 2cm from the well is 2000bp as interpolated from the standard curve

21. Fig. 10. 0-1. Restriction enzyme gel
Fig Restriction enzyme gel. An 8% agarose gel loaded with a 10kb ladder and phage DNA. Each lane labeled according to its content

22. Virtual Digestion Gels
Seaphages.org
MathBench

23. Common Challenges To Expect
Low DNA yield and quality
Loading problems
Dilution problems
Miscast gels
Pipetting errors