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(Multi Use Spin Filters)
Quantity: 100 Filters
(Price $99.00)
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| Multiple
use of Spin Columns include: |
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Isolate DNA from
agarose slice. |
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Exchange buffers rapidly. |
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Remove primers from PCR reactions. |
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Remove excess labels/ nucleotides
in random or end labeling. |
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Principle:
Quick Spin columns contain gel filtration matrix which
allow large molecules (e.g., DNA or RNA) to pass through
quickly by molecular sieving while retaining small contaminants
such as salts and small nucleotides. |
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| Protocol
#1 |
Protocol for the extraction
of DNA fragments from high melt agarose gels.
(This
protocol does not require gel filtration matrix)
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1. Excise
the gel slice from a high melt agarose containing the
DNA band of interest. Slice gel into small pieces and
place it inside the spin filter. Insert the spin filter
into receiving tube. This protocol is suitable for high
melt agarose gel only, do not use low melt agarose. We
also recommend using 0.5 X TAE buffer for agarose gel
electrophoresis.
 
Optional step: In order to increase the recovery
of DNA, place the spin filter (containing your gel slice)
at -80 degree centigrade for at least 10 minutes. However,
you can keep it at –80 degree centigrade for indefinite
period of time which will not result in loss of quality
of DNA.
2. Quickly, centrifuge for 5 minutes
at maximum speed (12-14,000 g).
3. Receiving tube will contains
eluted DNA which can be used directly for subsequent reactions
such as labeling, DNA ligation, PCR, fill-in/kinase reaction
with the Klenow fragment or four deoxynucleotides and
rATP sequencing reactions etc.,
Note: Recovery of DNA is usually between 60 to
80% - depends upon size and concentration of DNA. You
may verify the DNA concentration by using small aliquot
of eluted DNA and run it on a gel.
If you are planning to use eluted DNA for DNA ligation,
it is recommended that you run the gel electrophoresis
by using 0.5X TAE as running buffer. After extracting
the DNA from gel slice, add an equal amount of water to
the eluted DNA from the spin filter. Salt concentration
will be low enough and will not interfere with subsequent
ligation reactions.
If you wish to remove TAE buffer completely or to concentrate
DNA, follow protocol # 2. Do not directly concentrate
eluted DNA (from agarose gel slices) with ethanol as
any trace of agarose will co-precipitate with your DNA
which will make it difficult to resuspend your precipitated
DNA.
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| Protocol
#2 |
Purification of DNA
and removal of salt or unused primers or dNTP's from the
PCR/restriction enzyme products.
This protocol is applicable to following:
» Remove salts from enzymatic reaction
products.
» Remove unused primers, dNTP's from
the PCR prior to cycle sequencing
» Remove
excess labels/ nucleotides from random or end labeling
reactions.
» Remove salts/buffers
from eluted agarose gel slices (from protocol #1).
A: Hydrated Sephadex G-50: Weigh about 1 gram
of Sephadex G-50 and suspend it in about 25 ml of sterile
water. Sephadex is fully swelled in about 5 hours or leave
overnight at room temperature. Once Sephadex is fully
swelled, store it at 4 degree centigrade for further use.
It is recommended that you make few aliquots of hydrated
gel in separate tubes for further use. Hydrated gel with
sufficient amount of sterile water can be stored at 4
degrees C for about 6 months.
B: Loading gel into spin column and removal of excess
fluid:
1. By using a pipet, remove most of water from
the Sephadex containing tube. This should leave you with
thick slurry of Sephadex G-50. Add ~700 µl of Sephadex
G-50 slurry into a spin column. Insert spin column into
a receiving tube.
2. Make sure that the centrifuge is fully balanced.
Spin for two minutes @ 2000 RPM and remove the excess
fluid from the receiving tube.
Optional: In order to tightly pack the column,
you can refill the gel with additional Sephadex G-50
slurry and spin once again for two minutes @ 2000 RPM.
3. Insert spin filter back into receiving
tube and transfer 20 to 50µl of the sample to
the center of gel matrix without disturbing the Sephadex
bed surface. Centrifuge for 3 minutes @ 3000 RPM. Store
eluted DNA at -20 degree centigrade for subsequent uses.
Note: Care must be taken to place the sample directly
in the middle of the Sephadex gel surface of the column.
Touching the sides of the column may reduce the separation
efficiency. Load the sample within 10 minutes to avoid
drying of the gel.
Optional: Small amount of extracted DNA can be analyzed
by agarose gel electrophoresis to verify the quantity
of eluted DNA. If necessary, the remainder of the sample
can be concentrated by ethanol precipitation. In order
to concentrate the DNA, add 1/10 volume of 3 M sodium
acetate and 3 volumes of 100% ethanol. Keep the solution
in ice or at -20 degree for 10 minutes. To precipitate
the DNA, Spin samples for 10 minutes at maximum speed
by using a cold microfuge. Remove traces of ethanol
by using a clean pipette tip and resuspend the DNA pellet
in an appropriate amount of water or TE. |
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