User:Bryan Barney/Notebook/Microsatellite PCR development/2010/02/02

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Cancer magister microsatellites Main project page

Optimizing microsatellite PCR

Purpose

To perform agarose gel electrophoresis on PCR results on the following loci: COI, Cma103, Cma114, and Cma118
NOTE:
COI PCR performed by Caib Fainslow on Cma from the Monterey, CA area.
PCR of microsatellite loci Cma103, Cma114, and Cma 118 performed by Bryan Barney

Materials

100ml erlenmeyer flask
Agarose
TAE buffer
EtBr

Protocol

  1. Create a 2% agarose gel by adding 1g of agarose and 50ml of 1X TAE buffer to an erlenmeyer flask
  2. microwave for ~1 minute or until agarose is fully dissolved.
  3. allow the agarose to cool slightly (down to 65°C or so)
  4. add 5μL of 10% EtBr and swirl the flask to mix
  5. pour gel, allow to harden at room temperature (or at 2-8°C if in a hurry)
  6. Load Gel
    1. lane 1: 100bp Ladder 5μL (Promega)
    2. all samples: 2μL of sample, 7μL water, 1μL 10X loading dye
  7. Run gel at 50V for 5 minutes, then 100V for 45 minutes
  8. Visualize gel on GelDoc and take picture for file

Results

NOTE: - while loading ladder onto gel #2, the ladder sample floated up out of the gel to the surface of the TATE buffer. The same TAE buffer was used to make BOTH gels and load BOTH gel boxes, and the same ladder was loaded onto both gels - why the sample floated in gel 2 and not in gel 1 is a mystery!

Gel #1 Image:Geldocxr_2010-02-2.jpg

Gel #2 Image:Geldocxr_2010-02-3.jpg

Discussion -- lack of good bands


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