Chelex Extraction of Muddy hair Samples

adapted by Hannah Dugdale from Petra Carpenter’s methodology

Preparing Hair Sample

Will need: Samples stored in 80% ethanol (to kill M. bovis; 80% ETOH can also contain 100umol/l EDTA to keep DNA happy), ddH2O, 1.5 ml eppendorfs, scissors and tweezers, glass plate, presept (2 tablets in 500ml ddH2O), 4 glass beakers, squirty bottle of ddH2O, 10% bleach, tissue, plastic bag, marker pen.

  1. Label a 1.5ml eppendorf for your sample and add 1ml ddH2O
  2. Divide the glass plate into 8 sections with a marker pen, and place a hair sample on the first section (use a different section for each sample). Using tweezers separate 20 guard hairs with intact follicles.
  3. Hold the 20 hairs in tweezers over a beaker and squirt ddH2O to remove mud.
  4. Cut bottom 2cm of the hair (with root) off into the eppendorf, and discard of the remainder in a plastic bag.
  5. Leave to stand at room temperature for 30mins to remove the ethanol from the sample. Can then store samples in fridge over night before going onto next stage.
  6. Clean tweezers and scissors in ddH2O, then presept, then ddH2O and dry on tissue before using on a different sample. Clean gloves with 10% bleach in-between samples.

Extract ion of hair samples

Will need: centrifuge, 56°C rotating oven, 95°C heatblock, ddH2O, 5% autoclaved Chelex, (2.5g Chelex powder in 50ml low TE [10mM Tris pH8, 0.01mM EDTA]) magnetic stirrer, 10% bleach

Use 10% bleach to sterilise working surface before commencing extraction.

Include an extraction negative (can contain hair without root).

1.  Place 5% Chelex on magnetic stirrer to get Chelex into solution.

2.  Spin eppendorfs for 3 mins at 13000rpm, then remove all of the supernatant.

3.  Add 1ml ddH2O, vortex for 10-15 secs, then remove all of the supernatant. Repeat until you can’t see any mud left in the tube.

4.  Add 200ul of 5% Chelex

5.  Put tubes in-between 2 eppendorf racks and tip by hand for 2 mins to mix.

6.  Incubate in rotating oven at 56°C for 45 mins

7.  Tip tubes, held together between 2 racks, for 2 mins to mix

8.  Boil at 95°C for 8 mins (check roots are completely immersed before boiling – pulse spin if needed). Be careful when taking tubes off hotblock as the lids may pop open.

9.  Tip tubes, held together between 2 racks, for 2 mins to mix

10.  Spin 3 mins at 13000rpm

11.  Remove supernatant into a labelled sterile tube, being extremely careful not to take up any chelex beads – avoid the beads!