gel elution of 6KB PCR product

Fathi Hassan via methods%40net.bio.net (by fhassan69 from yahoo.com)
Mon Dec 27 05:12:16 EST 2010


hello,

you can try to clone the PCR product you got from gel in any cloning vector like 

pJet or pGem, since they require low amounts of PCR products, then you can 
digest the vector and get the fragment ready for further cloning. it is two 
steps more but may help you
good luck
FH



________________________________
From: DK <dk from no.email.thankstospam.net>
To: methods from magpie.bio.indiana.edu
Sent: Sun, December 26, 2010 12:28:15 AM
Subject: Re: gel elution of 6KB PCR product

In article <mailman.445.1293295295.15153.methods from net.bio.net>, "B.Rama chandran" 

<chandranbrama from gmail.com> wrote:
>Dear All,
>
>       I am facing problem in the  gel elution of 6KB PCR product, I am
>getting very less yield (~10ng/micto liter). I am using  Sigma gel
>extraction kit (catalog no:NA1111). Since yield is very less I couldn't use
>that product for restriction digestion. If you have any suggestion please
>give me. If you know any other technique which will give better yield please
>let me know that.

What is the yield as % of input? If you didn't have much to begin 
with, nothing can help. ~50% on gel-extraction are ~ normal. If it is the 
yield that is poor, try the same protocol with 2 kbp. If the result is good, 
your kit is not appropriate for 6K. If the result is bad, either your 
reagents are gone bad/improperly prepared or you are doing something 
wrong. I've purified 14 kbp from gel with about 30% using Qiagen's 
kit. Never a problem. 

DK
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<html><head><style type="text/css"><!-- DIV {margin:0px;} 
--></style></head><body><div 
style="font-family:arial,helvetica,sans-serif;font-size:12pt">you can try to 
clone the PCR product you got from gel in any cloning vector like pJet or pGem, 
since they requier low amounts of PCR products, then you can digest the vector 
and get the fragment ready for further cloning. it is two steps more but may 
help you<br>good luck<br>FH<br><br><div style="font-family: 
arial,helvetica,sans-serif; font-size: 12pt;"><div style="font-family: 
arial,helvetica,sans-serif; font-size: 12pt;"><font face="Tahoma" size="2"><hr 
size="1"><b><span style="font-weight: bold;">From:</span></b> DK 
&lt;dk from no.email.thankstospam.net&gt;<br><b><span style="font-weight: 
bold;">To:</span></b> methods from magpie.bio.indiana.edu<br><b><span 
style="font-weight: bold;">Sent:</span></b> Sun, December 26, 2010 12:28:15 
AM<br><b><span style="font-weight: bold;">Subject:</span></b> Re: gel elution of
6KB PCR product<br></font><br>
In article &lt;<a 
ymailto="mailto:mailman.445.1293295295.15153.methods from net.bio.net" 
href="mailto:mailman.445.1293295295.15153.methods from net.bio.net">mailman.445.1293295295.15153.methods from net.bio.net</a>&gt;,
 "B.Rama chandran" &lt;<a ymailto="mailto:chandranbrama from gmail.com" 
href="mailto:chandranbrama from gmail.com">chandranbrama from gmail.com</a>&gt; 
wrote:<br>&gt;Dear All,<br>&gt;<br>&gt;&nbsp; &nbsp; &nbsp;  I am facing problem 
in the&nbsp; gel elution of 6KB PCR product, I am<br>&gt;getting very less yield 
(~10ng/micto liter). I am using&nbsp; Sigma gel<br>&gt;extraction kit (catalog 
no:NA1111). Since yield is very less I couldn't use<br>&gt;that product for 
restriction digestion. If you have any suggestion please<br>&gt;give me. If you 
know any other technique which will give better yield please<br>&gt;let me know 
that.<br><br>What is the yield as % of input? If you didn't have much to begin 
<br>with, nothing can help. ~50% on gel-extraction are ~ normal. If it
is the <br>yield that is poor, try the same protocol with 2 kbp. If the result 
is good, <br>your kit is not appropriate for 6K. If the result is bad, either 
your <br>reagents are gone bad/improperly prepared or you are doing something 
<br>wrong. I've purified 14 kbp from gel with about 30% using Qiagen's <br>kit. 
Never a problem. 
<br><br>DK<br>_______________________________________________<br>Methods mailing 
list<br><a ymailto="mailto:Methods from net.bio.net" 
href="mailto:Methods from net.bio.net">Methods from net.bio.net</a><br><span><a 
target="_blank" 
href="http://www.bio.net/biomail/listinfo/methods">http://www.bio.net/biomail/listinfo/methods</a></span><br></div></div>

</div><br>

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