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

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

(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)

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

Digestion Reaction Setup: 1. First, gently mix DNA tubes, then incubate @ 65ᴼC for 10 minutes and then quickly place on ice. Gently mix DNA tubes, then incubate @ 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 tube @ 37ᴼC for 1 hour

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

General Overview Relaxed phage DNA 65oC, ~5 mins

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 tube @ 37ᴼC for up to 1 hour

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

General Overview Load samples into wells with the loading dye Remember to change tips between each sample Cover, connect electrodes to power supply Run current @120V until samples are out of the wells then @ 80V for about 45 to 60 minutes

Fig. 10. 0-1. Restriction enzyme gel Fig. 10.0-1. Restriction enzyme gel. An 8% agarose gel loaded with a 10kb ladder and phage DNA. Each lane labeled according to its content

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

DNA Ladder Fragment Size (bp) Standard Curve Table 10.4-1. Sample data table for creating DNA standard curve for agarose gel shown in Figure 10.4-1 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 10.4-1. Sample restriction enzyme gel.

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

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

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

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

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

Fig. 10. 0-1. Restriction enzyme gel Fig. 10.0-1. Restriction enzyme gel. An 8% agarose gel loaded with a 10kb ladder and phage DNA. Each lane labeled according to its content

Virtual Digestion Gels Seaphages.org http://phageenzymestools.com MathBench program @mathbench.umd.edu Nebcutterv2.0 @labtools.us/nebcutter-v2-0

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