Luckau Protocols:Agarose Gel: Difference between revisions

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# Remove combs
# Remove combs
* Pre-poured gels may be stored covered in the fridge for up to 1 week
* Pre-poured gels may be stored covered in the fridge for up to 1 week


===Load Agarose Gel===
===Load Agarose Gel===
# In titer tray, mix sample with gel loading buffer in 1:6 ratio
#* if loading 6µL into gel, mix 1µL gel loading buffer with 5µL sample in titer tray
# Transfer from titer tray into gel wells


===Run Agarose Gel===
# Attach rig's safe lid and ensure power supply leads fit snugly
# Plug loose ends of lead into power supply
#* black gets plugged into the ground
#* red gets plugged into the red
# Turn power supply to 'on'
# Adjust voltage according to table
#* for most applications 120V should be sufficient
# Check for the curtain of small bubbles to ensure everything is working properly
#





Revision as of 12:56, 19 January 2011

Agarose Gel Protocol

Tara K. Luckau's Home Page

Conservation Genetics Lab Notebook

Tara's Protocols

Purpose

Agarose gels are used to verify the presence and estimate the size of DNA (both genomic and amplified). When loaded into a gel and subjected to an electric current, DNA fragments migrate to the positive terminal. Small DNA fragments migrate more rapidly than large ones. By running a commercially available ladder of fragments of known size, we can estimate the DNA fragment size in our sample of interest.

To visualize the DNA after electrophoresis, a dye called Gel Red is added during the agarose-making process. The dye binds to the DNA and flouresces when exposed to UV light.

Materials To Be Familiar With

  • Gel Rig, caster tray, combs

Protocol

  • Use 1% for genomic DNA, 2% for amplified DNA
Gel Rig Size 1x TAE (mL) 1.5% agarose (g) 2% agarose (g) Gel Red (µL) Sample volume (µL) max voltage (V)
small (10cm x 10cm) 50 _ 1 5 4-6 80
medium (x x) 130 _ 2.6 13 24-tooth comb: 6-10 120
36-tooth comb: 3-5
large (20cm x 27cm) 270 _ 5.4 27 24-tooth comb: 6-10 170
36-tooth comb: 3-5


Pour Agarose Gel

  1. According to the table above, mix agarose powder and 1x TAE in Erlenmeyer flask; swirl
  2. Microwave on 1/2 power until the mixture starts to boil
    • Note: be careful not to let the mixture boil over, or you'll lose much of your volume and will have a mess to clean up
    • Note: make sure all the agarose has dissolved into solution
  3. Add GelRed when agarose mixture is warm; swirl
  4. Allow to cool - do the baby milk test (it should be at a warm temperature, but not too warm for a baby to touch)
  5. Pour into caster; set combs; allow to harden (10-20 minutes); will appear cloudy
  6. Remove combs
  • Pre-poured gels may be stored covered in the fridge for up to 1 week


Load Agarose Gel

  1. In titer tray, mix sample with gel loading buffer in 1:6 ratio
    • if loading 6µL into gel, mix 1µL gel loading buffer with 5µL sample in titer tray
  2. Transfer from titer tray into gel wells


Run Agarose Gel

  1. Attach rig's safe lid and ensure power supply leads fit snugly
  2. Plug loose ends of lead into power supply
    • black gets plugged into the ground
    • red gets plugged into the red
  3. Turn power supply to 'on'
  4. Adjust voltage according to table
    • for most applications 120V should be sufficient
  5. Check for the curtain of small bubbles to ensure everything is working properly


FYI:

  • Migration of gel loading dyes Bromophenol Blue (175bp) and Xylene Cyanol FF (1500bp)