Adapted for Teladorsagia circumcincta exsheathed by Tiggy Grillo from:
Williams BD, Schrank B, Huynh C, Shownkeen R, Waterston RH (1992) A Genetic Mapping System in Caenorhabditis elegans Based on Polymorphic Sequence-Tagged Sites. Genetics 131, 609-624.
I only made a couple of changes from Tiggy's method for T. tenuis: I don't homogenise the worm, and I extended the incubation time from 30 min to overnight.
50
mM KCl
10mM
Tris pH 8.4
25mM
MgCl2
0.45%
Tween 20 or Triton X100
0.45%
NP40 or Igepal
60
ug/ml Proteinase K in dH20
For
10 mls of buffer ready for storeage minus proteinase K:
500
ul
1 M KCl
100
ul
1 M Tris pH 8
1
ml
25 mM MgCl2
45
ul
Triton X100* or Tween 20*
45
ul
Igepal*
Make
up to 10 ml with AR H20
Store
at -20 degC in 1 ml aliquots. Can be refrozen (as long as proteinase
K hasn't been added). Before use add 6 ul 10 mg/ml proteinase K (or 3
ul of 20 mg/ul) to 1 ml.
*Very
viscous so use a cut p200 filter tip
Add
20 ul WLB (including proK) to each well of 96-well PCR plate.
Freeze
-70 degC 10 min.
Incubate
at 65degC
overnight.
Boil
for 10 min.
Stored
at -20 degC.
Prepare
a new 20x dilution plate: add 1 ul xtr to 20 ul AR H2O.
Take
1 ul for 10 ul PCR. The final PCR contains 1/200 of the lysed worm.
Store
at -20 degC.