Click the back button on your browser window to return to the Weisblat Lab website
<br>
		<p>
		

		<h2>Chemical Devitellinization Protocol </h2>
		(Paul Chang '97)
		<ol>
			<li>Prepare 20 ml of 0.1% trypsin in 18 mM CaCl2 HTR, pH 8.2 </li>
			<li>Aliquot into 20, 1 ml eppendorf tubes; store aliquots in -20c freezer until use </li>
			<li>Immediately before use, make sure solution is fully thawed, and at room temperature; add 10 mM DTT to each aliquot (0.0015 g per 1 ml)</li> 
			<li>Prepare 3 agarose covered petri dishes </li>
			<li>Fill one with devitellinization solution; fill other two with 18 mM CaCl2 HTR penicllin/streptomycin/gentamycin solution for washing </li>
			<li>Flame 4 Pasteur pipettes to blunt sharp edges </li>
			<li>Place embryos in devitellinization solution </li>
			<li>Watch embryos carefully; when vitelline envelope begins to lift off embryo and break open, transfer embryo into wash solution</li> 
			<li>Each embryo will "break" at different times, treat each as an individual </li>
			<li>As soon as all embryos are in wash, transfer to new wash dish </li>
			<li>Wait 5 minutes between washes </li>
		</ol>




		<h2>Manual Devitellinization Protocol</h2> 
		(Bob Goldstein '98)

		<h3>Method #1: Using Insect Pins</h3>

		Fixed embryos can be manually devitellinized one at a time with insect pins or more quickly in bulk with a narrow-bore pipet.  The narrow-bore pipet method will damage some embryos (<10% of the embryos when done well).  Damaged embryos are discarded and the remainder are used.<br>

		<h3>Method #2: Using Pasteur Pipettes</h3>
		<ol>
			<li>Prepare a narrow-bore pipet by pulling the ends of 5-10 glass pasteur pipets over a flame:</li>
			<li>Using watchmakers' forceps, cut the end of each pipette to a diameter roughly just smaller than the diameter of vitelline envelopes: Under the dissecting scope, place pipette next to some embryos (for size comparison). Start by cutting at a diameter a bit too small, and then work the way up the pipette a little at a time, breaking off pieces until the opening is the right diameter.  Jagged edges are okay.</li>
			<li>After wash, fixed embryos can then be devitellinized by pulling and pushing them into and out of the narrow bore pipet, one batch at a time.  Make sure the dish has plenty of liquid or air will make loads of bubbles.  Embryos will get stuck and/or damaged in pipets with bores that are too narrow, and will not be devitellinized in pipettes with bores that are too large.  Rinse and re-use the best narrow bore pipet of the 5-10 made and discard the rest.</li>
		<ol>

		 
		</p>