Bio-Rad#166-0007EDU:
Forensic DNA Fingerprinting Checklist
Name:__________________________________________Date:_________________
Please, check off each item upon completion !!!!!
Each group: 1- styrofoam coffee cup ½ filled w/ crushed ice.
1-clear 1.5mL microtube
6- colored microtubes
I. Restriction Digest. Kit designed for 8 groups.
_______1) Pass out six colored microtubes to each group & Label each colored tube with the
indicated abbreviations (in chart below) & place in foam micro test tube holder.
Green
Blue
Orange
Purple(Violet) Pink(Red)
Yellow
Crime Scene DNA
(CS)
Suspect 1
(S1)
Suspect 2
(S2)
Suspect 3
(S3)
Suspect 4
(S4)
Suspect 5
(S5)
Enzyme: 10 µL
10 µL
10 µL
10 µL
10 µL
10 µL
DNA: 10 µL
10 µL
10 µL
10 µL
10 µL
10 µL
________2) To each group, distribute one clear 1.5 mL microtube and label the tube: ENZ
________3) Aliquot 70 µL of enzyme mix into clear 1.5 mL microtubes for each group
(Note: You only need 60 but will have 10 extra microliters).
Put on ice if possible. Try using small Styrofoam coffee cup w/ crushed ice.
_______4) Which enzymes are in the mix: ______________ & ________________
_______5) Pipet 10 µL of enzyme mix (ENZ) into each colored tube listed above. **Do you
need to change the tip each time?_____________
_______6) The stock solutions of DNA will be rotated in order around the room. Pipet 10 µL of
each into the corresponding colored micro tube. Pipet up and down to mix well! **Do you
need to change the tip each time? ________________
_______8) Tightly cap tubes and mix components by flicking & then tapping on table. If
microfuge is available, pulse-spin to collect all liquid in the bottom of tube.
_______9) Place tubes in foam holder and incubate in water bath for 45 min at 37oC.
_______10) When time is up, place in refrigerator until agarose gels are ready.
Time to make agarose gels so we can see the results of our digests!!!
Princeton Satellite Site
Spring 2010
K. McKone
Bio-Rad#166-0007EDU:
Forensic DNA Fingerprinting Checklist
II. Preparing 1% Agarose Gels
Needed: 50x TAE Buffer  1x
Agarose Powder
1 - 250 mL flask/group; 2 - 500 mL flask/class
Hot Plates or Microwave
Graduated Cylinders or 50 mL Conical Tubes
_______1) Determine the volume of the gel casting tray by pouring a measured volume of
water from a graduated cyclinder into the tray.
Volume = ______________mL
_______2) Buffer Dilution of TAE (Tris-acetate-EDTA) Buffer: The buffer is at a
concentration of 50x. Each group needs to prepare 200 mL w/ a concentration of 1x.
One group can prepare the total for the class ( if 5 groups = 1,000 mL)
50x ( ? ml of concentrate) = 1x (1000 mL)
_________mL of 50x Buffer + __________mL distilled water = _______ mL of 1x Buffer
_______3) Prepare 1% agarose gel w/ a volume of 30 mL using 1x Buffer:
a) Each group uses their 250 mL flask to obtain 30 mL of TAE buffer from the class solution.
b) Determine amount of agarose powder needed for 30 mL of a 1% gel:
Agarose Powder = _________________g
_______4) Add measured agarose powder to buffer.
_______5) Boil the mixture above. Let it cool to about 65oC (It’s still warm but you can hold in your hand
w/out discomfort.) Don’t let it cool too much before pouring……Problems???
_______6) Before or immediately after pouring, place 8 tooth comb into the appropriate slot of
the gel tray.
______7) Clean your flask before gel particles start to form!
_______8) Allow gels to solidify at room temp for 10 - 20 minutes. They will appear cloudy
(opaque) when ready to use. You may use this time to clean glassware and empty waste
beaker. (May be a good time to practice loading gels if practice gels are available.)
_______9) Some protocols suggest pouring buffer over the gel before removing comb. Carefully
remove the comb from the solidified gel.
Princeton Satellite Site
Spring 2010
K. McKone
Bio-Rad#166-0007EDU:
Forensic DNA Fingerprinting Checklist
III. Loading Gels
Each Group:
1-little eppie (0.65mL microtube)
5 μL Loading Dye/sample or 30 μL /group
OMIT 1) Omit unless planning to do quantitative analysis of DNA fragments. ONE PERSON
will PREPARE HindIII Lambda Digest (DNA Marker): Heat marker to 65oC for 5
minutes, then chill on ice. (Results in better separation of marker).
_______2) Student: __________________ passes out a “little” eppie to each group for
Loading Dye. Label this tube “LD”.
_______3) Student: ___________________ aliquots ________μL into Loading Dye
tube/group.
_______4) Obtain your digested samples from refrigerator.
_______5) Using a new tip each time, add 5 μL of loading dye to your DNA sample tubes.
Green
Blue
Orange
Purple(Violet) Pink(Red)
Yellow
Crime Scene DNA
(CS)
Suspect 1
(S1)
5 μL
Suspect 2
(S2)
5 μL
Suspect 3
(S3)
5 μL
Suspect 5
(S5)
5 μL
Loading Dye: 5 μL
Suspect 4
(S4)
5 μL
_______6) Flick each tube to mix and Tap to bring contents to bottom of tube.
_______7) Pour electrophoresis buffer over gel if this has not already been done ~ 175 mL (volume
depends on gel box being used). Buffer should just cover the wells by 1-2 mm.
OMIT 8) Obtain tube of HindIII lambda digest (DNA marker). Make sure loading dye has already
been added.
_______9) Load DNA samples into wells of gel in the PROPER order. Gels may be read from left to right
(right side closest to hinge). Notice designated volumes for each sample (Reduce the volume loaded if
necessary so wells won’t overflow). CHANGE TIPS EACH TIME!
Lane 1
HindIII
10 μL
Loaded by:
2
CS (green)
20 μL
3
S1
20 μL
4
S2
20 μL
5
S3
20 μL
6
S4
20 μL
7
S5
20 μL
8
Blank
IV. Gel Electrophoresis
_______1) Close lid on gel box; make sure the color leads match. Connect electrical leads to power
supply.
_______2) Turn on power supply: 100 V for 30 minutes (75 V for ~ 45 minutes). Use your own
judgement; run longer if necessary. Better band separation the farther DNA runs.
Princeton Satellite Site
Spring 2010
K. McKone
Bio-Rad#166-0007EDU:
Forensic DNA Fingerprinting Checklist
V. Stain and Analyze Gel
Needed: 500x Fast Blast & staining trays
2-500 ml Flask/Class
1-250 ml Flask/Group
*200ml/group tap water (40-550C)
Fast Blast DNA stain (500x) should be diluted to 100x. For overnight staining, use 1x.
_______1) Each group needs ~ 150 mL of 100x Fast Blast unless they can share trays.
______ ml of 500x Fast Blast + _______ ml dH2O = 150 ml of 100x stain
Stain Gels
________2) Remove gel from casting tray and slide into stain.. (May try 2 gels/staining tray).
Pour ~ 150 mL of stain into staining tray to completely submerge gels.
________3) Stain for 2-3 minutes but not more than 3 minutes (Darker stain requires more destain
time). Use a funnel to transfer stain to original container when finished.
 We will save the stain. It can be reused at least 7 times.
Rinse Cycle
________4) Transfer gels into another container w/ 200 mL of clean, warm (40-55oC) tap
water. Gently shake the gel in the water for ~ 10 seconds to rinse.
________5) Wash again by performing step #4 but for 5 minutes. Gently rock the water once
every 20 seconds.
________6) Perform another wash, same as step #5 for 5 minutes.
________7) Pour off the water (into designated sink) and examine gels. Bands will be fuzzy but
begin to sharpen within 5-15 minutes.
________8) Place gel on white light (if possible).
________9) Sketch bands below:
Lane 1
HindIII/Lambda
2
CS
3
S1
4
S2
5
S3
6
S4
7
S5
8
Blank
23,130 bp
9,416
6,557
4,361
2,322
2.027
*Which suspect’s DNA was found at the crime scene? _________________
*If correct…Congratulations!! We will forward your name to CSI: Miami.
Princeton Satellite Site
Spring 2010
K. McKone