DOC

Student Guide to DNA Fingerprinting by PCR

By Tom Ray,2014-12-23 12:42
13 views 0
Student Guide to DNA Fingerprinting by PCR

    Student Guide to DNA Fingerprinting by PCR

    Exercise 1: Isolation of Cheek Cell DNA

    Introduction: In this exercise, each student will isolate DNA from his or her cheek cells (“mouthwash DNA”); this DNA will later be amplified by the polymerase chain reaction (PCR). Cheek cells will first be collected, by rinsing out your mouth with a saline solution (the salt keeps cells in proper osmotic balance so they don’t burst). They are concentrated by spinning in a centrifuge, and then boiled with a resin (Chelex). During the boiling, cells are disrupted and the DNA (now in single-stranded form) extracted in water. The Chelex removes impurities that would otherwise interfere with PCR.

Equipment:

    Clinical centrifuge, micro centrifuge, 1000μl pipettes, thermal cycler, boiling water

    bath with distilled water, floater.

    Supplies: 15ml conical centrifuge tubes, paper cup, 1.5μl micro centrifuge tubes (two for each reaction), blue pipette tips, ice bucket, gloves and forceps.

    Reagents: 10ml of 0.9%(w/v) saline (NaCl) in distilled water; 10% Chelex suspension (ion-exchange resin).

    A note on cheek cells: Once cells have been collected into saline, it is important to spin and concentrate them as soon as possible before they start to burst. We will work with only 6 samples at a time, which fit into the clinical centrifuge. Once these are concentrated, we can start collecting the next set of 6 samples.

STEP INSTRUCTIONS VISUAL

    Write your initials CLEARLY on the STEP

    15ml centrifuge tube containing ONE:

    sterile saline, on the paper cup, and Tip:

    on two 1.5μl micro centrifuge tubes Use all three Sterile Saline 15 ml centrifuge initials on (label tops). sides and top of micro centrifuge Tip: KEEP ON ICE AT ALL TIMES!! 2- 1.5 μl micro centrifuge tubes tube.

Student Guide to DNA Fingerprinting by PCRb HELWIG 1

    Pour all of the saline solution into STEP

    your mouth, slosh it around vigorously TWO:

    for 10 seconds, and expel it into the Tip

     paper cup. Slosh your Crimp one side to make a spout on the Dixie mouth side to Only 6 people can go at a time. Do not start sloshing cup. Side, very until the centrifuge reads 1 minute. Slosh until you vigorously. Do are ready to place your centrifuge tube directly not eat, drink into the centrifuge.

    OR chew gum,

    preferably 1 Why saline?

    hour before The salt keeps the cheek cells in proper osmotic lab. balance so they don’t burst. This is an isotonic

    state. IV’s are .9 saline solutions also for proper

    osmotic balance.

    Pour the solution back into the 15ml STEP

    centrifuge tube, and immediately THREE

    place it into the clinical centrifuge.

    Spin cells 10 minutes at maximum STEP

    speed in the centrifuge. FOUR:

    Fact: This is called

    cell

    fractionation.

    The reason for

    10 minutes is

    to suspend the

    nuclei.

    Being careful not to disturb the STEP

     pellet, pour the supernatant into the FIVE

    Tip: paper cup. Try not to tip the tube Try not to tip back and forth; this will re-suspend the tube back the pellet and lose cells. Remove as and forth; this will re-suspend much supernatant as possible; when the pellet and you notice the pellet starting to lose cells.

    loosen stop pouring. (Additional

    supernatant can be removed with a

    1000μl pipette.)

    Using a 1000μl pipette set at 500μl, STEP

    pipette the Chelex suspension up and SIX

    down to re-suspend the beads.

    Immediately pipette 500μl of Chelex Tip: Make