<!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN"> <html> <head> <meta content="text/html; charset=ISO-8859-1" http-equiv="content-type"> <title></title> </head> <body> <p class="MsoNormal" style="margin-left: -2.85pt; text-align: center;" align="center"><span style="font-size: 16pt;">cDNA Synthesis and Alexa dye labeling in Preparation for Microarray hybridization<o:p></o:p></span></p> <p class="MsoNormal" style="margin-left: -2.85pt; text-align: center;" align="center"><span style="font-size: 16pt;"><o:p>&nbsp;</o:p></span></p> <p class="MsoNormal" style="margin-left: -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin-left: 33.15pt; text-indent: -0.25in;"><!--[if !supportLists]--><b style=""><span style="">1)<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></b><!--[endif]--><b style="">Denaturation of RNA and annealing of primers:<o:p></o:p></b></p> <p class="MsoNormal" style="margin-left: -2.85pt;"><span style=""> </span><span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>15 <span style="font-family: Symbol;">µ</span>g total RNA</p> <p class="MsoNormal" style="margin-left: -2.85pt;"><span style=""> </span><span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>random 18-mer at 2 <span style="font-family: Symbol;">µ</span>g/<span style="font-family: Symbol;">µ</span>l, 4 <span style="font-family: Symbol;">µ</span>g=2<span style="font-family: Symbol;"> </span><span style="font-family: Symbol;">µ</span>l</p> <p class="MsoNormal" style="margin-left: -2.85pt;"><span style=""> </span><span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>random hexamers at 3 <span style="font-family: Symbol;">µ</span>g/<span style="font-family: Symbol;"> </span><span style="font-family: Symbol;">µ</span>l, 10 <span style="font-family: Symbol;">µ</span><span style="font-family: Symbol;"></span>g=3.3 <span style="font-family: Symbol;">µ</span>l</p> <p class="MsoNormal" style="margin-left: -2.85pt;"><span style=""> </span><span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>RNase-free ddw QS to 18 <span style="font-family: Symbol;">µ</span>l</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 38.85pt;"><b style="">Or<o:p></o:p></b></p> <p class="MsoNormal" style="margin-left: -2.85pt;"><span style=""> </span><span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>isolated mRNA from 10 <span style="font-family: Symbol;">µ</span><span style="font-family: Symbol;"></span>g of total RNA</p> <p class="MsoNormal" style="margin-left: -2.85pt;"><span style=""> </span><span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>random hexamers at 3 <span style="font-family: Symbol;">µ</span><span style="font-family: Symbol;"></span>g/<span style="font-family: Symbol;"> </span><span style="font-family: Symbol;">µ</span>l, 5 <span style="font-family: Symbol;">µ</span>g=1.6<span style="font-family: Symbol;">µ</span><span style="font-family: Symbol;"></span>l</p> <p class="MsoNormal" style="margin-left: -2.85pt;"><span style=""> </span><span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>RNase-free ddw QS to 18 <span style="font-family: Symbol;">µ</span><span style="font-family: Symbol;"></span>l</p> <p class="MsoNormal" style="margin-left: -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin-left: 0.5in; text-indent: 0.15pt;">Incubate each tube at 70<sup>o</sup>C for 5 min. to denature and then on ice for 1 minor more.<span style="">&nbsp; </span>Note that it is important to use the random 18-mer in addition to the random hexamers when starting with total RNA in order to favor priming of mRNA in the reaction.<span style="">&nbsp; </span>Also, I have found that the primer/RNA ratio stated above yields the highest amount of cDNA.</p> <p class="MsoNormal"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin-left: 33.15pt; text-indent: -0.25in;"><!--[if !supportLists]--><b style=""><span style="">2)<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></b><!--[endif]--><b style="">cDNA synthesis with amino-modified nucleotides: <o:p></o:p></b></p> <p class="MsoNormal" style="margin-left: -2.85pt;"><span style=""> </span><span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>With samples on ice, add to each tube:</p> <p class="MsoNormal" style="margin-left: -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">5X first strand buffer<span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>6.0 <span style="font-family: Symbol;">µ</span>l</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">0.1M DTT<span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>1.5 <span style="font-family: Symbol;">µ</span>l</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">dNTP mix<span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>1.5 <span style="font-family: Symbol;">µ</span>l</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">RNase out<span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>1.0 <span style="font-family: Symbol;">µ</span>l</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;"><u>Superscript RT<span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>2.0 </u><span style="font-family: Symbol;">µ</span><u><span style="font-family: Symbol;"></span>l<o:p></o:p></u></p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Final volume<span style="">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>30 <span style="font-family: Symbol;">µ</span>l</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin-left: 33.15pt;">Mix well, centrifuge briefly to collect contents at the bottom of the tube.<span style="">&nbsp; </span>Incubate tubes at 46<sup>o</sup>C for 2-3 hours.<span style="">&nbsp; </span>3 hour incubations will result in 20-30% higher cDNA yields.</p> <p class="MsoNormal"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin-left: 33.15pt; text-indent: -0.25in;"><!--[if !supportLists]--><b style=""><span style="">3)<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></b><!--[endif]--><b style="">Alkaline Hydrolysis of RNA and Neutralization:<o:p></o:p></b></p> <p class="MsoNormal" style="margin-left: -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Add 15 <span style="font-family: Symbol;">µ</span>l of 1N NaOH to each reaction tube.<span style="">&nbsp; </span>Mix thoroughly</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Incubate at 70<sup>o</sup>C for 10 min</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Neutralize with 15 <span style="font-family: Symbol;">µ</span>l of 1N HCL, mix gently</p> <p class="MsoNormal"><o:p>&nbsp;</o:p></p> <p class="MsoNormal"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin-left: 33.15pt; text-indent: -0.25in;"><!--[if !supportLists]--><b style=""><span style="">4)<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></b><!--[endif]--><b style="">Purification of cDNA using Qiagen PCR purification kit<o:p></o:p></b></p> <p class="MsoNormal" style="margin-left: 15.15pt;"><b style=""><o:p>&nbsp;</o:p></b></p> <p class="MsoNormal" style="text-indent: 33.15pt;">Add 300 <span style="font-family: Symbol;">µ</span>l of buffer PB to sample, mix, load onto Qiaquick column</p> <p class="MsoNormal" style="text-indent: 33.15pt;">Centrifuge at room temperature for 1 min</p> <p class="MsoNormal" style="text-indent: 33.15pt;">Discard flow through</p> <p class="MsoNormal" style="text-indent: 33.15pt;">Add 750 <span style="font-family: Symbol;">µ</span>l<span style="font-family: Symbol;"> </span>of wash buffer PE (make sure you ve added ethanol to the buffer)</p> <p class="MsoNormal" style="text-indent: 33.15pt;">Centrifuge at room temperature for 1 min</p> <p class="MsoNormal" style="text-indent: 33.15pt;">Discard flow through</p> <p class="MsoNormal" style="text-indent: 33.15pt;">Centrifuge for another min. to dry out the column</p> <p class="MsoNormal" style="text-indent: 33.15pt;">Move column to a new eppendorf tube</p> <p class="MsoNormal" style="text-indent: 33.15pt;">Apply 30 <span style="font-family: Symbol;">µ</span>l RNase-free sterile ddw to the column membrane</p> <p class="MsoNormal" style="text-indent: 33.15pt;">Allow to sit at room temperature for 1 min.</p> <p class="MsoNormal" style="text-indent: 33.15pt;">Centrifuge for 1 min to collect the cDNA</p> <p class="MsoNormal" style="margin-left: -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="text-indent: 33.15pt;">Quantitate the yield of cDNA by diluting 2 <span style="font-family: Symbol;">µ</span><span style="font-family: Symbol;"></span>l (+98 <span style="font-family: Symbol;">µ</span>l ddw), read A<sub>260<o:p></o:p></sub></p> <p class="MsoNormal" style="margin-left: 33.15pt;">Typical yields of cDNA starting from total RNA are around 10 <span style="font-family: Symbol;">µ</span>g and from mRNA-around 1 <span style="font-family: Symbol;">µ</span>g or more.</p> <p class="MsoNormal"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin-left: 33.15pt; text-indent: -0.25in;"><!--[if !supportLists]--><b style=""><span style="">5)<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></b><!--[endif]--><b style="">Coupling with Alexa dyes:<o:p></o:p></b></p> <p class="MsoNormal" style="margin-left: -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin-left: 33.15pt;">Dry cDNA in speedvac on medium heat setting until the sample is in 3 <span style="font-family: Symbol;">µ</span>l or less, <b style="">DO NOT</b> dry completely or the cDNA will be stuck to the tube and be very hard to resuspend (check every 5 min. or less).</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Add 5 <span style="font-family: Symbol;">µ</span>l 2X coupling buffer to each sample</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Add 2 <span style="font-family: Symbol;">µ</span>l <u>room temperature</u> DMSO to a vial of Alex Fluor to resuspend dye</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Add DMSO/dye solution to cDNA</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Incubate at room temperature in the dark for 1-2 hours</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Reaction can be stored overnight in the dark if needed</p> <p class="MsoNormal"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin-left: 33.15pt; text-indent: -0.25in; font-weight: bold;"><!--[if !supportLists]--><span style="">6)<span style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><!--[endif]-->Purification of Fluorescently labeled cDNA using Qiagen PCR purification kit</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Add 30 <span style="font-family: Symbol;">µ</span>l of buffer PB to sample, mix, load onto Qiaquick column</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Centrifuge at room temperature for 1 min</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Discard flow through</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Add 750 <span style="font-family: Symbol;">µ</span>l<span style="font-family: Symbol;"> </span>of wash buffer PE (make sure you ve added ethanol to the buffer)</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Centrifuge at room temperature for 1 min</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Discard flow through, there should be color on the column at this point</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Centrifuge for another min. to dry out the column</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Move column to a new eppendorf tube</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Apply 30 <span style="font-family: Symbol;">µ</span>l RNase-free sterile ddw to the column membrane</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Allow to sit at room temperature for 1 min.</p> <p class="MsoNormal" style="margin-left: 33.15pt;">Centrifuge for 1 min to collect the dye labeled cDNA, these should be vibrantly colored</p> <p class="MsoNormal" style="margin-left: -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Analyze 2 <span style="font-family: Symbol;">µ</span>l (+98 <span style="font-family: Symbol;">µ</span>l ddw) on the spec to determine yield</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">Scan samples from 240-800 nm and calculate the following:</p> <p class="MsoNormal" style="margin-left: -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">ng of total cDNA=(A<sub>260</sub>-A<sub>320</sub>) X 37ng/<span style="font-family: Symbol;">m</span>l X volume in <span style="font-family: Symbol;">m</span>l X dilution factor</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">pmoles Alexa Fluor 555=(A<sub>555</sub>-A<sub>650</sub>)/0.15 X volume in <span style="font-family: Symbol;">m</span>l X dilution factor</p> <p class="MsoNormal" style="margin-left: -2.85pt; text-indent: 0.5in;">pmoles Alexa Fluor 647=(A<sub>650</sub>-A<sub>750</sub>)/0.24 X volume in <span style="font-family: Symbol;">m</span>l X dilution factor</p> <p class="MsoNormal" style="margin-left: -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt 33.15pt;">Base/dye ratio for Fluor 555={(A<sub>260</sub>-A<sub>320</sub>)-[(A<sub>555</sub>-A<sub>650</sub>) X 0.04]} X 150,000/(A<sub>555</sub>-A<sub>650</sub>) X 6,600</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt 33.15pt;">Base/dye ratio for Fluor 647={(A<sub>260</sub>-A<sub>320</sub>)-[(A<sub>650</sub>-A<sub>750</sub>) X 0]} X 239,000/(A<sub>650</sub>-A<sub>750</sub>) X 6,600</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt; text-indent: 0.5in;">The number of dye molecules per 100 bases is calculated:</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt; text-indent: 0.5in;">100/(base/dye ratio)</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt 33.15pt;">You can <u>minimally</u> expect <u>&gt;</u>24 pmole of incorporated dye and <u>&gt;</u>2.5 dye molecules/100 bases.<span style="">&nbsp; </span>I typically get around 900-1000 pmoles of dye incorporation when starting with total RNA and around 150 pmoles of dye incorporation for mRNA and 3.5-5 molecules of dye/100 bases.</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt 33.15pt;">Typically the cDNA from total RNA averages 500 bp in length as determined by gel electrophoresis with a range of ~1kb to ~200 bp relative to 100bp standard ladder.</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt; text-indent: 0.5in;">Random Hexamers=Invitrogen cat# 48190-011, 3 <span style="font-family: Symbol;">µ</span>g/ul</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt 33.15pt;">Tandom 18-mer=Invitrogen custom oligo synthesis 50 nM synthesis $7.20, quote #508786</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;"><b style="">Cautions</b>:</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;">The DMSO needs to be at room temperature before you open the tube so that it does not absorb moisture from the air which will prevent it from working properly.</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;">When adding ddw to the Qiagen membrane for elution, make sure the water is added to the surface of the membrane and not caught up on the sides of the column, otherwise the yield will be decreased.</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;">It is important to plan your experiment such that you know what array hybridizations you will be performing at the same time and what samples are to be compared directly so that you will know what RNAs and cDNAs will need to be prepared at the same time.<span style="">&nbsp; </span>It will not be possible to compare samples prepped on different days</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;">.</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;">You should have minimally 50 pmoles of each dye in an array hyb.</p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;"><o:p>&nbsp;</o:p></p> <p class="MsoNormal" style="margin: 0in -27pt 0.0001pt -2.85pt;">Always check the yield of cDNA in each reaction before proceeding with the dye labeling reaction.<span style="">&nbsp; </span>If you do not have the expected cDNA yield you will need to trouble shoot the first part of this procedure.</p> <p class="MsoNormal"><o:p>&nbsp;</o:p></p> </body> </html>