76
FENG QIAN AND GREGORY G. GERMINO
Low melting point
(LMP) agarose
Freeze-squeeze
Standard protocol
ever, and is usually used to isolate large fragments (>5 kb) which are inefficiently recovered by other methods.
A far simpler approach is to simply melt the gel slice containing the fragment
of interest. Standard preparations of agarose, however, require boiling to
melt the gel. This process denatures DNA and makes it unsuitable for many
subsequent manipulations. This problern is circumvented by the use of a
specially formulated agarose that melts at 65°C. There are a number of additional advantages of using this matrix. Since it remains fluid at 37°C and is
very low in contaminants, enzymatic manipulations (such as ligation, labeling, restriction digestion, etc.) can be performed by adding a portion of the
melted gel slice directlyto the reaction mix. Furthermore, the gentle nature of
the extraction procedure allows the isolation of intact high molecular weight
DNA (though it works equally weil for all sizes of DNA fragments).
An alternative method that is quick, inexpensive and efficient for recovery
of molecules up to 5 kb in length can be used to isolate fragments from
standard agarose gels. After excision, the gel slice is snap frozen in liquid
nitrogen, thawed and then squeezed, expressing the buffer which contains
the DNA.
1. Cut out the desired fragment from an agarose gel.
2. Punch a hole in the bottom of an microcentrifuge tube with a needle.
3. Layer glass wool in the bottom of the tube. The glass wool should cover
the hole.
4. After closing the cap, place the entire tubein liquid nitrogen for 30 sec to
freeze the agarose slice within the tube.
5. Place the frozen tube into a collection microcentrifuge tube which is put
below the frozen tube.
6. Centrifuge the entire device at 12,000 g for 5 min. The expressed liquid in
the collection tube contains the DNA molecules, which are ready for
ethanol precipitation.
Spin columns Amicon offers a range of spin columns that can be used to separate larger
DNA fragments from much smaller ones (i.e., >500 bp from <100 bp).
These have the advantage ofbeing very efficient, rapid, and useful for concentrating samples. The columns are especially useful for purifying PCR
fragments from the amplification mix of primers, enzyme, buffer and nucleotides.
FENG QIAN AND GREGORY G. GERMINO
Low melting point
(LMP) agarose
Freeze-squeeze
Standard protocol
ever, and is usually used to isolate large fragments (>5 kb) which are inefficiently recovered by other methods.
A far simpler approach is to simply melt the gel slice containing the fragment
of interest. Standard preparations of agarose, however, require boiling to
melt the gel. This process denatures DNA and makes it unsuitable for many
subsequent manipulations. This problern is circumvented by the use of a
specially formulated agarose that melts at 65°C. There are a number of additional advantages of using this matrix. Since it remains fluid at 37°C and is
very low in contaminants, enzymatic manipulations (such as ligation, labeling, restriction digestion, etc.) can be performed by adding a portion of the
melted gel slice directlyto the reaction mix. Furthermore, the gentle nature of
the extraction procedure allows the isolation of intact high molecular weight
DNA (though it works equally weil for all sizes of DNA fragments).
An alternative method that is quick, inexpensive and efficient for recovery
of molecules up to 5 kb in length can be used to isolate fragments from
standard agarose gels. After excision, the gel slice is snap frozen in liquid
nitrogen, thawed and then squeezed, expressing the buffer which contains
the DNA.
1. Cut out the desired fragment from an agarose gel.
2. Punch a hole in the bottom of an microcentrifuge tube with a needle.
3. Layer glass wool in the bottom of the tube. The glass wool should cover
the hole.
4. After closing the cap, place the entire tubein liquid nitrogen for 30 sec to
freeze the agarose slice within the tube.
5. Place the frozen tube into a collection microcentrifuge tube which is put
below the frozen tube.
6. Centrifuge the entire device at 12,000 g for 5 min. The expressed liquid in
the collection tube contains the DNA molecules, which are ready for
ethanol precipitation.
Spin columns Amicon offers a range of spin columns that can be used to separate larger
DNA fragments from much smaller ones (i.e., >500 bp from <100 bp).
These have the advantage ofbeing very efficient, rapid, and useful for concentrating samples. The columns are especially useful for purifying PCR
fragments from the amplification mix of primers, enzyme, buffer and nucleotides.