In article <ger-1608971931420001 at b461-powmac7500.stanford.edu>,
ger at stanford.edu (Gerard Manning) wrote:
> I've heard that it's possible to stain embryos with Xgal without fixation,
> in order select homozygous mutants away from blue balanced siblings, for
> PCR sequencing. Does anybody have any details of how it can be done? (I
> haven't yet tried, but I would imagine that it would require
> permeabilisation or removal of the vitelline membrane).
>> -Gerard Manning.
I have successfully PCRd DNA from homozygote embryos marked by the absence
of a "Blue balancer" (in my case TM3, ftz-Bgal). I basically followed the
method of Rastelli (EMBO J. 12, 1513-1522) to stain. Breifly,
Collect eggs 4-6 hrs old
Dechorionate in 50% bleach for 2 mins
Rinse with PBS
Shake vigorously in 50% heptane 50% PBS (to permeabilise the vitelline membrane)
Rinse 3x PBS + 0.3%Triton (until embyos don't clump together)
Rinse 2x Xgal staining buffer (855ul H2O, 40ul 0.1M K4Fe(CN)6, 40ul 0.1M
K3Fe(CN)6, 20ul 10% Triton X, 23.5ul 0.2M Na2HPO4, 1.5ul 0.2M NaH2PO4)
Stain in staining buffer + 60ul/ml of 20mg/ml X-gal solution 1.5hours at 37C.
I pick the non-stained embryos (checking that they have developed and are
not unfertilized). The staining paterns are not very good but sufficient
to pick unstained embryos.
I then perform PCR reactions according to Garozzo and Christiansen (DIN
Single embryos are squashed in 10ul Gloor and Engel's buffer (10mM Tris pH
8.2, 1mM EDTA, 25mM NaCl, 200ug/ml proteinase K) using a yellow tip.
Incubate at 37C for 30 mins and 95C 2mins.
1ul is used for 50ul PCR reaction. I use 20pmols of each primer and 2.0 or
2.5mM MgCl2, 0.2mM dNTPs in Boehringer or Promega buffer and Taq.
I have directly sequenced the PCR products on an ABI sequencer (using the
same primers as for PCR) after gel purification and "Gene Cleaning" the
Of course this would be even easier if there were GFP balancers out there
Ian JH Roberts.