Week 1 of PCR
DNA Extraction/ Amplification of VNTR regions
Module 1: Isolation of DNA from Human Hair
1. Isolate 3-4 hairs containing a sheath (often white in color) encircling the shaft near the
base of the hair. This is easiest to do with eyebrow hair (pluck using tweezers and
mirror).
2. Place the hairs in the bottom of a 1.5 ml tube.
3. Obtain lysis solution from the front bench.
4. Mix the lysis solution by vortexing. Before it can settle, remove 150 μl and add it to
the tube containing the hair.
5. Make sure the sheaths are completely submerged in solution and are not stuck on the
sides of the tubes.
6. Place the tube in a 560 C waterbath for 15 minutes.
7. Remove the tube from the waterbath and allow it to cool for 30 seconds.
8. Vortex the tube for 15 seconds.
9. Check again that the hair sheaths are completely submerged in solution and are not
stuck on the sides of the tube.
10. Place tube in a float and place in boiling water for 10 minutes.
11. Remove tube from water and cool on ice for 2 minutes.
12. Vortex for 10 seconds.
13. Spin in a microcentrifuge for 30 seconds.
14. Carefully remove 50 μl of supernatant (fluid on top) and transfer it to a clean 1.5 ml
microcentrifuge tube. ***If you stir up the bottom and mix it at ALL, you must recentrifuge and try again.***
15. Place the tube with the supernatant on ice (this is the NEW tube you just filled, not
the old tube with the eyebrows in it).
Module II: Amplification of the D1S80 Locus
1. Label the tube containing the PCR reaction pellet with your initials (First, middle and
last please). Think about what needs to be in the pellet to allow this PCR to work!
2. Tap the reaction tube to assure the reaction pellet is at the bottom of the tube.
Prepare the reactions for PCR:
a. To prepare the tube for the PCR reaction mix, add the following to the
bead:
 D1S80 primer mix (freezer)
20.0 μl
 Hair DNA (supernatant on ice)
5.0 μl
b. Gently mix the PCR reaction tube (by flicking) to collect all the sample
at the bottom of the tube.
Make sure the PCR bead is completely dissolved.
3. Instructors will prepare a few PCR control reactions that will be used to compare to
our results (these will be the crime scene samples).
4. Students will bring their tubes to the instructor who will put the samples in a
programmed thermal cycler set at the following:
Initial Denaturation
940C for 5 min.
32 cycles @
940C for 30 sec
550C for 30 sec
720C for 30 sec
Final Extension
720C for 4 min.
5. Instructors will add 5 μl of 10x Gel Loading Solution to the samples and freeze until
the following week.