<HTML> <HEAD> <META NAME="GENERATOR" CONTENT="Adobe PageMill 2.0 Mac"> <TITLE>Hybridization of the probe to chromosomes.</TITLE> </HEAD> <BODY BGCOLOR="#edd7e0" BACKGROUND="../../Images/yrule.gif"> <P ALIGN=CENTER><IMG SRC=http://www.ksu.edu/wgrc/Images/tbar.gif WIDTH="550" HEIGHT="32" ALIGN="BOTTOM" NATURALSIZEFLAG="3"></P> <H1 ALIGN=CENTER><TT>Biotin labeling and hybridization of probe to chromosomes</TT></H1> <P><HR ALIGN=LEFT></P> <BLOCKQUOTE> <BLOCKQUOTE> <H4>Suggested chemicals: Nick Translation Kit, Enzo Diagnostics Cat. No. 42803.</H4> </BLOCKQUOTE> </BLOCKQUOTE> <H3>Biotin labeling of plasmid or genomic DNA.</H3> <DL> <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Remove slides from -70 C freezer, remove the cover slips with a razor blade, and dry the slides in an ethanol series (70 %, 95 %, 100 % ethanol; 5 min each at room temperature). Slides should be dried for several hours before ISH. <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Prepare the following hybridization mixture: <DL> <DD><TABLE WIDTH="276" HEIGHT="142" BORDER="2" CELLSPACING="0" CELLPADDING= "0"> <TR> <TD WIDTH="79%" HEIGHT="18"> deionized formamide</TD> <TD WIDTH="21%"> <P ALIGN=CENTER> 10 µl</TD></TR> <TR> <TD HEIGHT="18"> 20 X SSC</TD> <TD> <P ALIGN=CENTER> 2 µg</TD></TR> <TR> <TD HEIGHT="20"> sheared salmon sperm DNA (10 mg/ml)</TD> <TD> <P ALIGN=CENTER> 2 µl</TD></TR> <TR> <TD HEIGHT="18"> probe DNA</TD> <TD> <P ALIGN=CENTER> 0.5-2 µl</TD></TR> <TR> <TD HEIGHT="17"> Deionized or distilled water</TD> <TD> <P ALIGN=CENTER> X µl</TD></TR> <TR> <TD HEIGHT="18"> 50 % dextran sulfate</TD> <TD> <P ALIGN=CENTER>4 µl </TD></TR> <TR> <TD HEIGHT="30"> Total reaction volume (use H2O to adjust the total volume to 20 µl)</TD> <TD> <P ALIGN=CENTER> 20 µl</TD></TR> </TABLE> </DL> </DL> <BLOCKQUOTE> <DL> <DD>10 µl of the hybridization mix is used for one slide and covered with a '18 x 18' cover slip. Care should be taken that the sloution is well mixed, because the 50 % dextran sulfate solution is very sticky. </DL> </BLOCKQUOTE> <DL> <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> For genomic ISH, prepare the hybridization mixture as below: <DL> <DD><TABLE WIDTH="274" HEIGHT="129" BORDER="2" CELLSPACING="0" CELLPADDING= "0"> <TR> <TD WIDTH="79%" HEIGHT="18"> deionized formamide</TD> <TD WIDTH="21%"> <P ALIGN=CENTER> 10 µl</TD></TR> <TR> <TD HEIGHT="17"> 20 X SSC</TD> <TD> <P ALIGN=CENTER> 2 µg</TD></TR> <TR> <TD HEIGHT="19"> sheared salmon sperm DNA (10 mg/ml)</TD> <TD> <P ALIGN=CENTER> 2 µl</TD></TR> <TR> <TD HEIGHT="17"> probe DNA</TD> <TD> <P ALIGN=CENTER> 1 µl</TD></TR> <TR> <TD HEIGHT="17"> blocking DNA</TD> <TD> <P ALIGN=CENTER> 1 µl</TD></TR> <TR> <TD HEIGHT="17"> 50 % dextran sulfate</TD> <TD> <P ALIGN=CENTER>4 µl </TD></TR> <TR> <TD HEIGHT="21"> Total reaction volume</TD> <TD> <P ALIGN=CENTER> 20 µl</TD></TR> </TABLE> </DL> </DL> <BLOCKQUOTE> <DL> <DD>The amount of the sheared blocking DNA is the most critical part of this procedure. The blocking DNA should be in excess of 100 to300 times more than the probe DNA. </DL> </BLOCKQUOTE> <H3>Hybridization of probe to chromosomes.</H3> <DL> <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Denature the mixture by placing at 75 C for 5 min, immediately chilling on ice for 5 min, and spining down the solution. <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Place the slides (by using a slide holder that can handle 10-20 slides) in 70 % formamide in 2 X SSC (140 ml formamide + 20 ml 20 X SSC + 40 ml H2O, this solution can be used several times; the quality of the formamide is important, i. e., formamide from CMS is of constant good quality) at 70 C for 2 min. <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Immediately transfer the slides into an ethanol series (70 %, 95 %, and 100 %, 5 min each at 20 C), and air dry the slides. <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Add 10 µl of the hybridization mixture to each slide and cover with an '18 x 18' mm cover slip. Place the slides in a moist chamber (any kind of box can be used with 2 layers of wet, 3 mm Whatman paper on the bottom; use plastic bars to hold the slides). <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Incubate the slides in the moist chamber at 37 C for a minimum of 6 h or overnight. </DL> <P><HR ALIGN=LEFT></P> <P ALIGN=CENTER><IMG SRC=http://www.ksu.edu/wgrc/Images/bbar.gif WIDTH="550" HEIGHT="52" ALIGN="BOTTOM" NATURALSIZEFLAG="3"></P> <P ALIGN=CENTER><A HREF=http://www.ksu.edu/><FONT SIZE=-1>Home</FONT></A><FONT SIZE=-1> | <A HREF=http://www.ksu.edu/search/>Search</A> | <A HREF=http://www.ksu.edu/aboutuni/new/>What's New</A> | <A HREF=http://www.ksu.edu/aboutuni/help/>Help</A> | <A HREF=http://www.ksu.edu/aboutuni/comment-form.cgi>Comments</A> <BR> <A HREF=http://www.ksu.edu/>Kansas State University</A> | <A HREF=http://www.ksu.edu/wgrc/>Wheat Genetics Resource Center</A> <BR> 31 January, 1997.</FONT> </BODY> </HTML>