Restriction Analysis of Plasmid DNA

Session 1: Restriction Digest Reactions

Your sample is EITHER plasmid DNA pAMP or pKAN. Which do you have? How could you tell them apart? What’s different between them?

Each restriction enzyme cuts DNA wherever its recognition site appears. Each restriction enzyme recognizes a particular sequence of nucleotides, called its restriction site. Many recognition sites are palindromes. BamHI …NNNGGATCCNNN… …NNNG GATCCNNN… …NNNCCTAGGNNN… …NNNCCTAG GNNN… HindIII …NNNAAGCTTNNN… …NNNA AGCTTNNN… …NNNTTCGAANNN… …NNNTTCGA ANNN…

A restriction map identifies where restriction sites appear along a DNA. BamHI cuts here HindIII cuts here What will be different between the DNA fragments produced by cutting pAMP vs. pKAN with BamHI & HindIII?

DNAs can be distinguished from each other by restriction mapping. 3755 bp 2332 bp 784 bp 1875 bp 1904 – 1120 = 784 4539 – 784 = 3755

Glove Up! Put on a pair of lab gloves S, M, L, XL available Most hands will fit in M or L gloves. Try those sizes first unless you have particularly small or large hands. Made of nitrile (no latex = no allergies)

Micropipetting Review 1st Step, 1st Stop. 2nd Step, 2nd Stop. Micropipetting Review FIRST STEP: Measuring SECOND STEP: Dispensing Choose a micropipette whose range spans the desired volume. Set the micropipette to the desired volume by turning the plunger knob. Don’t force past the pipette’s limits, as this breaks the pipette! Place a tip on the micropipette, matching tip and plunger colors. Depress plunger to the FIRST STOP and HOLD. Place the tip into the liquid. Slowly release the plunger, keeping tip in liquid. Place tip against side of tube, near bottom. Depress plunger to the SECOND STOP and HOLD. Remove tip from tube while holding down plunger. Release plunger. Throw away used tip using the ejector button. Use a new tip each time you pipet.

Label a Restriction Digest Tube From the jar with the white screw cap, remove one 1.5ml microtube. With a lab marker, label the lid of the microtube with your period number and the first initials of each team member. P1 TDH

Add Plasmid DNA Your team was given a sample of either pAMP or pKAN plasmid DNA in a tube labeled “DNA” and a number between 1 and 12. From this tube, use your micropipette to measure 5μl (microliters) of plasmid DNA and transfer it to your Restriction Digest tube. At 0.1μg/μl, this 5μl contains 0.5μg or 500ng of DNA. DNA 1…12 5μl P1 TDH

Add Water From the tube labeled H2O, measure 9μl of water and transfer it to your Restriction Digest tube. H2O 9μl P1 TDH

Add Restriction Reaction Buffer Enzymes require a chemical environment of the right pH and concentration of ions. The 5X restriction buffer is a concentrated mix that provides the environment needed for the restriction enzymes to work properly. From the tube labeled 5X RE Buffer, measure 4μl of 5x Restriction Digest Buffer and transfer it to your Restriction Digest tube. 5X RE Buffer 4μl P1 TDH

Add Restriction Enzymes You will cut your plasmid DNA with two restriction enzymes: BamHI and HindIII. From the tube labeled BamHI + HindIII measure 2μl of the BamHI and HindIII mix and transfer it to your Restriction Digest tube. BamHI + HindIII 2μl P1 TDH

Incubate the Restriction Digest Reaction Close the cap on your Restriction Digest tube and place it in the heating block set at 37°C. The restriction enzymes work best at 37°C. The reactions will incubate for one hour, then be stored in a freezer until you examine them using gel electrophoresis.

Prepare the Restriction Digest Reactions Agarose Gel Electrophoresis 4/12/2018 Prepare the Restriction Digest Reactions Reaction Component Volume to Add Your Plasmid DNA Sample ( 5µl H2O 9µl 5X RE Buffer 4µl BamHI + HindIII Restriction Enzyme mix 2µl Total Volume 20µl dNTP mix from Pitt Stockroom: 200ul @ 10mM for $58 -> dilute 1:5 to 2mM (add 800ul H2O) -> makes 80 x 12.5ul (2.5-reaction) aliquots

Session 2: Gel Electrophoresis

Prepare Your Samples for Loading Agarose Gel Electrophoresis 4/12/2018 Prepare Your Samples for Loading Add 4µl of the 6X Loading Dye to your restriction digest sample. If your liquids are sticking separately to the side of the tube, flick the tube with your finger and tap the bottom gently on your lab bench, or spin briefly in microcentrifuge to collect entire sample at bottom of tube.

Load Your Sample On The FlashGel When called, bring to the FlashGel: Your DNA sample Micropipette with tip Load 6μl of your sample into a well.

Agarose Gel Electrophoresis 4/12/2018 Write your team initials or team number below the well into which you loaded your sample. Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 1kb ladder Period ____ Teacher: Print out copies of this slide to place near each gel each period. Have students write their initials or team number in the lane into which their sample was loaded.

Run the Gel A power supply provides current to the electrodes and through the buffer and gel. The progress of migration through the gel is monitored with tracking dyes that are visible without the transilluminator. 1.2% Flash Gel 200 V 8 minutes

Analysis of Gel Results

Restriction Mapping Can Be Used To Identify Unknown DNAs 3755 bp 2332 bp 784 bp 1875 bp

Agarose Gel Electrophoresis 4/12/2018 1kb ladder 1 2 3 4 5 6 7 8 9 10 11 12 13 Restriction Fragment Sizes pAMP: 3755, 784 pKAN: 2332 1875 Promega BenchTop 1kb Ladder Period #1 Teacher: After capturing a digital image with the Flash Gel software, return to this slide. Right-click on the gel image, choose “Change Picture…”, and browse to the gel image you just captured. That will replace this image with your new one. 1.2% 200V 8min

What Questions Do YOU Have?

Materials

Agarose Gel Electrophoresis 4/12/2018 100bp DNA Markers Bio-Rad EZ Load 100bp Molecular Ruler #170-8352; $99 Ready to load Invitrogen 100 bp DNA Ladder #15628-019; $99 In TE Invitrogen TrackIt 100 bp DNA Ladder #10488-058; $114 Promega 100bp DNA Ladder #G2101; $111 -20C RT 10 bands 100 – 1000 bp 16 bands 100 – 1500bp + 2072bp 600bp band 2x-3x brighter 11 bands 100-1000bp + 1500bp 500bp band brighter 1 µg/µl 100ng/µl x 5ul/lane = 500ng/lane 130ng/ul x 5ul/lane = 650ng/lane 50ug 500ul = 50ug = 100 lanes 250ul = 32.5ug = 50 lanes These are the 100bp DNA ladders that are stocked in the Department of Biological Sciences stockroom.

Agarose Gel Electrophoresis 4/12/2018 1kb DNA Markers Bio-Rad EZ Load 1kp Molecular Ruler # #170-8355; $84 Ready to load Invitrogen 1kb DNA Ladder #15615-016; $158 In TE Invitrogen TrackIt 1 Kb DNA Ladder #10488-072, $98 Ready-to-load Invitrogen TrackIt 1 Kb Plus DNA Ladder #10488-085, $125 Promega 1kp DNA Ladder #G5711; $111 -20C RT 15 bands 1 – 15kb 22 bands 1 – 12kb + 100bp-500bp 20 bands 0.1 – 12kb Doublet 1650/2000 1 µg/µl 100ng/µl x 5ul/lane = 500ng/lane 100ng/ul x 5ul/lane = 250 ul = 250ug 500ul = 50ug = 100 lanes These are the 1kb DNA ladders that are stocked in the Department of Biological Sciences stockroom.

Teacher Pre-Lab Preparation and Classroom Set-Up

FastDigest HindIII: 3 μl Preparing DNA Samples “DNA A” 1kb Ladder 200ng / 5μl 2-3 stations “DNA B” Lambda/HindIII 500ng / 5μl 2 stations “DNA C” 100ng / 5μl “DNA D” pAMP/BamHI/HindIII “DNA E” pKAN/BamHI/HindIII FlashGel: “load 5-20ng/band in 5ul/well” We load 100ng/5ul/lane 3 lanes/gel 1 gel/period 6 periods/day Prepare 3.0 μg Stock Conc.100 ng/μl pAMP DNA: 30 μl H2O: 18 μl 10X FastDigest Bfr: 6μl FastDigest BamHI: 3 μl FastDigest HindIII: 3 μl Final Rxn. Vol. = 60 μl 37° 30 min; 37° 5 min Add 90 μl 1X TE Final Conc. = 20 ng/μl Load 100 ng = 5 μl (Flash Gel recommends 5 μl / well)

Lambda () / HindIII DNA Markers Bacteriophage Lambda’s genome is 48,502 bp. When cut with the restriction enzyme HindIII, eight fragments are released. /HindIII is a commonly used set of DNA size standards for electrophoresis. Which DNA sample is lambda/HindIII?

Plotting a Standard Curve for the DNA Markers Plot on Semi-Log Paper X-axis = distance migrated Y- axis = DNA length in base pairs (bp)

Workstation Set-Up

Supplies Boxes of S, M, L, X nitrile lab gloves 0.5ml microcentrifuge tubes Bottle of water for gels Styrofoam cups ice

Equipment Microcentrifuges Vortex mixers FlashGel running tray with blue-light illuminator Power supply FlashGel camera Laptop computer with FlashGel software Traditional electrophoresis tank with