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Date: Dec 28, 2010 3:00 AM
Subject: bionet.molbio.methds-reagnts - 2 new messages in 2 topics - digest
To: "bionet.molbio.methds-reagnts digest subscribers" <bionet.molbio.methds-reagnts@googlegroups.com>
bionet.molbio.methds-reagnts
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Today's topics:
* Methods Digest, Vol 67, Issue 8 - 1 messages, 1 author
http://groups.google.com/group/bionet.molbio.methds-reagnts/t/ca739b039a1c5a1f?hl=en
* gel elution of 6KB PCR product - 1 messages, 1 author
http://groups.google.com/group/bionet.molbio.methds-reagnts/t/fcdaf69d6c779d50?hl=en
==============================================================================
TOPIC: Methods Digest, Vol 67, Issue 8
http://groups.google.com/group/bionet.molbio.methds-reagnts/t/ca739b039a1c5a1f?hl=en
==============================================================================
== 1 of 1 ==
Date: Sun, Dec 26 2010 5:35 am
From: Virash Gupta
Dear B Ram,
try following following method. make a very small hole at the bottom
of a 0.5 ml PCR tube and cover it with some sterile glass wool. Put
your cut gel band in this tube and close it. Now put this tube into a
1.5 ml eppendorf tube. using an appropriate balancing tube spin at
12,000 rpm for 5 min. you should get 70-80% of your DNA in the
eppendorf tube which can be precipitated and resuspended in small
volume. Alternatively, put this tube into 1.5 ml tube containing ~100
microlitre TAE buffer. Also add same amount of this buffer in 0.5 ml
tube. Take two small pieces of platinum wires, dip one through space
between 0.5 ml and 1.5 ml tube so that it immerses in the buffer.
other piece of platinum wire should be purt in 0.5 ml tube buffer.
apply low current (~ 10V) with black electrode connected to wire in
0.5 ml tube and red electrode to other one immersed in 1.5 ml tube
buffer. This will make a small electrophoresis coloumn. after around
20 min, whole of DNA will move into buffer of 1.5 ml tube. Disacrd 0.5
ml tube along with its contents. precipitate DNA from buffer of 1.5 ml
tube using sod acetate and cold ethanol. All the best.
On 12/25/10, methods-request@oat.bio.indiana.edu
<methods-request@oat.bio.indiana.edu> wrote:
> Send Methods mailing list submissions to
> methods@net.bio.net
>
> To subscribe or unsubscribe via the World Wide Web, visit
> http://www.bio.net/biomail/listinfo/methods
> or, via email, send a message with subject or body 'help' to
> methods-request@net.bio.net
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>
> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of Methods digest..."
>
>
> Today's Topics:
>
> 1. gel elution of 6KB PCR product (B.Rama chandran)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Sat, 25 Dec 2010 18:52:35 +0530
> From: "B.Rama chandran" <chandranbrama@gmail.com>
> Subject: gel elution of 6KB PCR product
> To: Methods@magpie.bio.indiana.edu
> Message-ID:
> <AANLkTikscKjcve5M+V_SpxhzymrfjbiQ39HRep0YQOCS@mail.gmail.com>
> Content-Type: text/plain; charset=UTF-8
>
> 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.
>
>
> ------------------------------
>
> _______________________________________________
> Methods mailing list
> Methods@net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>
> End of Methods Digest, Vol 67, Issue 8
> **************************************
>
--
Dr V K Gupta
Sr Microbiologist (Mol Biology)
IMBL, Department of Entomology
Pun. Agric. Univ., Ludhiana (Pb)-141004- India
M: 081465-55515
==============================================================================
TOPIC: gel elution of 6KB PCR product
http://groups.google.com/group/bionet.molbio.methds-reagnts/t/fcdaf69d6c779d50?hl=en
==============================================================================
== 1 of 1 ==
Date: Mon, Dec 27 2010 2:12 am
From: Fathi Hassan
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@no.email.thankstospam.net>
To: methods@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@net.bio.net>, "B.Rama chandran"
<chandranbrama@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
_______________________________________________
Methods mailing list
Methods@net.bio.net
http://www.bio.net/biomail/listinfo/methods
--0-1362310605-1293367365=:9174
Content-Type: text/html; charset=us-ascii
<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
<dk@no.email.thankstospam.net><br><b><span style="font-weight:
bold;">To:</span></b> methods@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@net.bio.net"
href="mailto:mailman.445.1293295295.15153.methods@net.bio.net">mailman.445.1293295295.15153.methods@net.bio.net</a>>,
"B.Rama chandran" <<a ymailto="mailto:chandranbrama@gmail.com"
href="mailto:chandranbrama@gmail.com">chandranbrama@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@net.bio.net"
href="mailto:Methods@net.bio.net">Methods@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>
</body></html>
--0-1362310605-1293367365=:9174--
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From: "bionet.molbio.methds-reagnts group" <noreply@googlegroups.com>
Date: Dec 28, 2010 3:00 AM
Subject: bionet.molbio.methds-reagnts - 2 new messages in 2 topics - digest
To: "bionet.molbio.methds-reagnts digest subscribers" <bionet.molbio.methds-reagnts@googlegroups.com>
bionet.molbio.methds-reagnts
http://groups.google.com/group/bionet.molbio.methds-reagnts?hl=en
bionet.molbio.methds-reagnts@googlegroups.com
Today's topics:
* Methods Digest, Vol 67, Issue 8 - 1 messages, 1 author
http://groups.google.com/group/bionet.molbio.methds-reagnts/t/ca739b039a1c5a1f?hl=en
* gel elution of 6KB PCR product - 1 messages, 1 author
http://groups.google.com/group/bionet.molbio.methds-reagnts/t/fcdaf69d6c779d50?hl=en
==============================================================================
TOPIC: Methods Digest, Vol 67, Issue 8
http://groups.google.com/group/bionet.molbio.methds-reagnts/t/ca739b039a1c5a1f?hl=en
==============================================================================
== 1 of 1 ==
Date: Sun, Dec 26 2010 5:35 am
From: Virash Gupta
Dear B Ram,
try following following method. make a very small hole at the bottom
of a 0.5 ml PCR tube and cover it with some sterile glass wool. Put
your cut gel band in this tube and close it. Now put this tube into a
1.5 ml eppendorf tube. using an appropriate balancing tube spin at
12,000 rpm for 5 min. you should get 70-80% of your DNA in the
eppendorf tube which can be precipitated and resuspended in small
volume. Alternatively, put this tube into 1.5 ml tube containing ~100
microlitre TAE buffer. Also add same amount of this buffer in 0.5 ml
tube. Take two small pieces of platinum wires, dip one through space
between 0.5 ml and 1.5 ml tube so that it immerses in the buffer.
other piece of platinum wire should be purt in 0.5 ml tube buffer.
apply low current (~ 10V) with black electrode connected to wire in
0.5 ml tube and red electrode to other one immersed in 1.5 ml tube
buffer. This will make a small electrophoresis coloumn. after around
20 min, whole of DNA will move into buffer of 1.5 ml tube. Disacrd 0.5
ml tube along with its contents. precipitate DNA from buffer of 1.5 ml
tube using sod acetate and cold ethanol. All the best.
On 12/25/10, methods-request@oat.bio.indiana.edu
<methods-request@oat.bio.indiana.edu> wrote:
> Send Methods mailing list submissions to
> methods@net.bio.net
>
> To subscribe or unsubscribe via the World Wide Web, visit
> http://www.bio.net/biomail/listinfo/methods
> or, via email, send a message with subject or body 'help' to
> methods-request@net.bio.net
>
> You can reach the person managing the list at
> methods-owner@net.bio.net
>
> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of Methods digest..."
>
>
> Today's Topics:
>
> 1. gel elution of 6KB PCR product (B.Rama chandran)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Sat, 25 Dec 2010 18:52:35 +0530
> From: "B.Rama chandran" <chandranbrama@gmail.com>
> Subject: gel elution of 6KB PCR product
> To: Methods@magpie.bio.indiana.edu
> Message-ID:
> <AANLkTikscKjcve5M+V_SpxhzymrfjbiQ39HRep0YQOCS@mail.gmail.com>
> Content-Type: text/plain; charset=UTF-8
>
> 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.
>
>
> ------------------------------
>
> _______________________________________________
> Methods mailing list
> Methods@net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>
> End of Methods Digest, Vol 67, Issue 8
> **************************************
>
--
Dr V K Gupta
Sr Microbiologist (Mol Biology)
IMBL, Department of Entomology
Pun. Agric. Univ., Ludhiana (Pb)-141004- India
M: 081465-55515
==============================================================================
TOPIC: gel elution of 6KB PCR product
http://groups.google.com/group/bionet.molbio.methds-reagnts/t/fcdaf69d6c779d50?hl=en
==============================================================================
== 1 of 1 ==
Date: Mon, Dec 27 2010 2:12 am
From: Fathi Hassan
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@no.email.thankstospam.net>
To: methods@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@net.bio.net>, "B.Rama chandran"
<chandranbrama@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
_______________________________________________
Methods mailing list
Methods@net.bio.net
http://www.bio.net/biomail/listinfo/methods
--0-1362310605-1293367365=:9174
Content-Type: text/html; charset=us-ascii
<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
<dk@no.email.thankstospam.net><br><b><span style="font-weight:
bold;">To:</span></b> methods@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@net.bio.net"
href="mailto:mailman.445.1293295295.15153.methods@net.bio.net">mailman.445.1293295295.15153.methods@net.bio.net</a>>,
"B.Rama chandran" <<a ymailto="mailto:chandranbrama@gmail.com"
href="mailto:chandranbrama@gmail.com">chandranbrama@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@net.bio.net"
href="mailto:Methods@net.bio.net">Methods@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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--0-1362310605-1293367365=:9174--
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