gel elution of 6KB PCR product

B.Rama chandran

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Dec 25, 2010, 8:22:35 AM12/25/10

to Met...@magpie.bio.indiana.edu

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.

regards,
B.Ram.

Message has been deleted

Fathi Hassan

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Dec 27, 2010, 5:12:16 AM12/27/10

to met...@oat.bio.indiana.edu

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 <d...@no.email.thankstospam.net>
To: met...@magpie.bio.indiana.edu
Sent: Sun, December 26, 2010 12:28:15 AM
Subject: Re: gel elution of 6KB PCR product

In article <mailman.445.129329...@net.bio.net>, "B.Rama chandran"

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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Methods mailing list
Met...@net.bio.net
http://www.bio.net/biomail/listinfo/methods

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Content-Type: text/html; charset=us-ascii

<html><head><style type="text/css"></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 good luck FH <div style="font-family:
arial,helvetica,sans-serif; font-size: 12pt;"><div style="font-family:
arial,helvetica,sans-serif; font-size: 12pt;"><hr
size="1">From: DK
<d...@no.email.thankstospam.net> To: met...@magpie.bio.indiana.edu Sent: Sun, December 26, 2010 12:28:15
AM Subject: Re: gel elution of
6KB PCR product 
In article <<a
ymailto="mailto:mailman.445.129329...@net.bio.net"
href="mailto:mailman.445.129329...@net.bio.net">mailman.445.129329...@net.bio.net</a>>,
"B.Rama chandran" <<a ymailto="mailto:chandr...@gmail.com"
href="mailto:chandr...@gmail.com">chandr...@gmail.com</a>>
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 _______________________________________________ Methods mailing
list <a ymailto="mailto:Met...@net.bio.net"
href="mailto:Met...@net.bio.net">Met...@net.bio.net</a> <a
target="_blank"
href="http://www.bio.net/biomail/listinfo/methods">http://www.bio.net/biomail/listinfo/methods</a> </div></div>

</div>

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Wedad Al Salmi

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Jan 3, 2011, 5:47:02 PM1/3/11

to d...@no.email.thankstospam.net, met...@magpie.bio.indiana.edu

Hello Everyone,

I was searching on the best method to purify large quantities of pure plasmid from a 250ml culture. I came across to this protocol by nature protocols (
http://www.nature.com/nprot/journal/v2/n5/full/nprot.2007.171.html) and was wondering how good it is. They report that once you make the ELP protein and its helper protein you will be able to purify up to 300ug of plasmid from 5ml culture in 30 minutes with high purity.

Also, where can I find ELP-MerR fusion protein (ELP153MR) and helper protein, ELP153H6. Currently I am in Washington DC.

And if anyone can advice me on what's the best method to purify large quantities of plasmid DNA, I would be appreciate it a lot.

Thank you,

Wadad Al Salmi