Methods Digest, Vol 67, Issue 8

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Sun Dec 26 08:35:47 EST 2010


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 from oat.bio.indiana.edu
<methods-request from oat.bio.indiana.edu> wrote:
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>    1. gel elution of 6KB PCR product (B.Rama chandran)
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> Message: 1
> Date: Sat, 25 Dec 2010 18:52:35 +0530
> From: "B.Rama chandran" <chandranbrama from gmail.com>
> Subject: gel elution of 6KB PCR product
> To: Methods from magpie.bio.indiana.edu
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> 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.
>
>
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-- 
Dr V K Gupta
Sr Microbiologist (Mol Biology)
IMBL, Department of Entomology
Pun. Agric. Univ., Ludhiana (Pb)-141004- India
M: 081465-55515



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