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