<HTML> <HEAD> <META NAME="GENERATOR" CONTENT="Adobe PageMill 2.0 Mac"> <TITLE>Preparation and purification of biotin-labeled probe.</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>Preparation and purification of biotin-labeled probe for ISH</TT></H1> <P><HR ALIGN=LEFT></P> <H3>I. Biotin-labeling of plasmid or genomic DNA.</H3> <H4>Suggested chemicals: Nick Translation Kit, Enzo Diagnostics Cat. No. 42803.</H4> <DL> <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Prepare the following solution <DL> <DD><TABLE WIDTH="267" HEIGHT="121" BORDER="2" CELLSPACING="0" CELLPADDING= "0"> <TR> <TD WIDTH="76%" HEIGHT="21"> dNTP (including biotin-11-dUTP</TD> <TD WIDTH="24%"> <P ALIGN=CENTER> 5 µl</TD></TR> <TR> <TD HEIGHT="20"> plasmid or genomic DNA</TD> <TD> <P ALIGN=CENTER> 1 µg</TD></TR> <TR> <TD HEIGHT="20"> DNA polymerase I</TD> <TD> <P ALIGN=CENTER> 5 µl</TD></TR> <TR> <TD HEIGHT="20"> Dnase I</TD> <TD> <P ALIGN=CENTER> X µl</TD></TR> <TR> <TD HEIGHT="18"> Deionized or distilled water</TD> <TD> <P ALIGN=CENTER> Y µl</TD></TR> <TR> <TD HEIGHT="19"> Total reaction volume</TD> <TD> <P ALIGN=CENTER> 50 µl</TD></TR> </TABLE> <DD>Use H2O to adjust the total volume to 50 µl, mix carefully after each step, and spin down at the end. </DL> <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Incubate the reaction tube in a water bath at 14-15 C for 2 h. <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Stop the reaction by adding 5 µl stop buffer. </DL> <P>The most critical part of the labeling, if the DNA is clean, is the size of the final nick translation product. The best ISH results are obtained when the size of the biotin-labeled probe is around 300-600 bp. To determine the size of the probe, denature 15 µl of the nick translation product in boiling water for 4 min and cool down on ice immediately; and electrophorese the sample on a mini-gel with a DNA size marker. The size of the nick translation product can be adjusted by adding a different amount of DNase I in the reaction solution. Random primer labeling also can be used for biotin-labeling, however the size of the probe is relatively easier to control by nick translation.</P> <H3>II. Purification of the biotin-labeled probe.</H3> <DL> <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Start 20 min before the end of the nick translation incubation. Plug the bottom of a 1 ml tuberculin syringe with siliconized glass wool. <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Fll the syringe to the top with Sephadex G-50 using a sterilized pasteur pipette. <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Place the Sephadex-filled syringe into a 15 ml Corex tube and centrifuge at 1,500 rpm for 4 min to pack down the Sephadex. <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Repeat the previous two steps until the packed Sephadex column has a total volume of about 0.9 ml. <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Wash the colum twice with 55 µl TE by spinning at 1,500 rpm for 4 min each. <DD><IMG SRC=http://www.ksu.edu/wgrc/Images/dot-yelw.gif WIDTH="14" HEIGHT="14" ALIGN= "BOTTOM" NATURALSIZEFLAG="3"> Place a sterilized 1.5 ml tube on the bottom of the 15 ml Corex tube and insert the packed and washed syringe into the Corex tube in such a way that the tip of the syringe fits into the 1.5 ml tube. Load the nick translation product (55 µl with stop buffer) and spin at 1,500 rpm for 4 min. The probe is collected in the 1.5 ml tube </DL> <P>The final volume of probe should be close to 55 µl. Usually, 5 µl of such a biotin-labeled probe (1 µg/55 µl = 18 ng/µl) is enough for one slide, and the probe can be used to process 100 slides. The size of the probe can be checked by electrophoresis on a mini-gel, either before or after the spin-colum purification. The probe can be stored in a freezer for several years.</P> <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>