Human Alu Insertion Polymorphism Experiment

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Human Alu Insertion Polymorphism Experiment Powered By Docstoc
					Detection of the human VNTR
using PCR*


*A Polymerase Chain Reaction Experiment
The experiment is divided into 2 parts

    Isolate human DNA from cheek cells
    The DNA will be amplified using PCR



    The DNA will be analyzed using gel electrophoresis
Polymerase Chain Reaction: another
    method of DNA amplification
    Basis of PCR

   Create conditions “in vitro” for DNA replication.
    Requirements for replication

   DNA template
   Primers
   Taq DNA Polymerase**
   Nucleotides
   MgCl++
Temperature cycles plays a key role:

   Unwinding DNA              98C
   Annealing                  45-60C
   Extension                  72C




    http://www.dnalc.org/Shockwave/pcranwhole.html
      Primers determine

   The sequence of DNA that will be amplified.
Basis for sequence specific amplification


 •Two primers are used to bracket the area you want to amplify.

 •Primers are single stranded synthetic sequences of DNA normally 20-30 bp.

 •One primer is complementary to the beginning of the target gene on one
 strand while the other primer is complementary to end of the target gene on
 the complementary strand.
Summary:

   Unwinding DNA              98C
   Annealing                  45-60C
   Extension                  72C




    http://www.dnalc.org/Shockwave/pcranwhole.html
The experiment is divided into 2 parts

    Isolate human DNA from cheek cells
    The DNA will be amplified using PCR



    The DNA will be analyzed using gel electrophoresis
Highlights of Key Steps of your DNA Isolation
Each person should obtain

   Cotton swabs

   A test tube with 2 mls of buffer

   A screw top microfuge tube

   2 transfer pipets
Isolation of DNA

     Cheek Cells

     Obtain cells by swabbing the inside of
      the mouth with a cotton tipped
      applicator.
Next…

   Place cotton head in 2 ml of PBS (in conical
    tube)

   Swirl vigorously for 1 min. to dislodge cells

   Press the cotton head against the walls to
    squeeze out as much liquid as possible

   Use new applicator and repeat the above
    steps.
Next…
   Transfer 2 ml to screw top tube.

   Spin at 5000 rpm for 1 min. Be sure you
    have pellet. If not repeat swabbing.

   Remove supernantant but SAVE PELLET
    (these are your cells!)

   Add 100 ul of chelating agent to sample
Next…

   Re-suspend the pellet in 100 ul of
    chelator solution: MAKE SURE
    PELLET IS RESUSPENDED!


   Place screw top microfuge tube in
    boiling waterbath for 10 minutes to
    break (lyse) cells.
Next:



     After 10 min boiling allow tube to
      cool (2 minutes and then spin
      microfuge tube for 2 minutes (5000
      rpm)
    Next:

   Obtain a PCR tube with “PCR bead”

   Add primers to bead and 5 ul of your
    supernatant and gently mix.

   Label your PCR tube your assigned seat
    number

   Place in PCR!
Summary of PCR tube


   To PCR tube containing “bead” add:

       20.0 ul of primer solution
       5.0 ul of cheek cell DNA
VNTR : PCR reaction 32 cycles at:

       94C                30 seconds
       65C                30 seconds
       72C                30 seconds
The gel electrophoresis

   Analysis of your results will be carried
    and provided to you (time may not be
    available at the next lab for gel
    electrophoresis).
        After PCR

   Gel electrophoresis
       Warm samples (including ladder) 2 minutes at 50
        C
       Load 25 ul of the sample
       Load 25 ul of ladder

				
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posted:7/27/2012
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