Insert Purification, Plasmid Ligation and Transformation
PLASMIDS FOR SUBCLONING
Add 1µg of plasmid (eg pBluescript KS+) to a microtube with a suitable
buffer (eg One?Phor-All+) in volume of 19.5µl. Add 0.5µl of
restriction enzyme(s) and incubate at 37°C for 2 hours.
Add 180µl of water, 100µl of chloroform and 100µl of
phenol. Vortex mix and spin for 2min at full speed.
Collect the supernatant in a fresh microtube and add 200µl of chloroform.
Vortex mix and spin for 2min at full speed.
Collect the supernatant in a fresh microtube and add 20µl of 3M NaOAc,
pH 7.0 and 500µl of ethanol. Mix and spin for 15 min. at full speed.
Pour off the supernatant, pulse spin the residual liquid down and draw
off with an autopippette, being careful not to disturb the (invisible)
pellet. Allow the pellet to dry for 5min at room temp by leaving the microtube
Resupend the pellet in 40µl of water. This is the cut plasmid at
a nominal concentration of 20ng/µl. 1µl of this is usually
sufficient for a ligation reaction.
It is not generally necessary to dephosphorylate cut plasmids for
subcloning. However for library production it is essential to minimise
plasmid religation and this method would not be applicable.
SUBCLONING RESTRICTION FRAGMENTS
Digest DNA of interest to generate fragment(s) for subcloning, at a concentration
which will allow easy detection of DNA in a 10µl volume after electrophoresis
(100-300ng of fragment).
At this stage the plasmid digests may be extracted, precipitated and combined
directly in a ligation reaction or they may be purified from agarose gels.
For gel purification, add 1.8g (for frags in range 750-5000bp, more agarose
for lower fragment sizes) of agarose, 0.5ml of 50X TAE buffer and 26ml
of water to a conical flask. Place a small conical flask, inverted, in
the neck of the larger flask and put into the microwave for 1min at full
power. Allow to cool, add 1.25µl of 10mg/ml ethidium bromide and
pour onto a minigel tray (7x10cm). Insert the comb (use 0.5cm wide wells)and
leave to set. Meanwhile make up (if not already in apparatus) 250ml of
TAE with 12.5µl of 10mg/ml ethidium bromide. Add 2µl of 6X
dye buffer to the restriction digest, assemble the gel in the apparatus,
add the buffer if required and load 10µl of the sample along side
suitable standards. Run at 60-80V until the bromophenol blue is about two
thirds of the way down. Visualise the band(s) of interest on a UV transilluminator
and excise in as small a gel volume as possible with a scalpel and weigh
into a microtube.
Add an equal volume of GFX gel extraction buffer (Pharmacia) and heat the
gel slice 60°C for 15mins, mixing intermittantly. Add to assembled
GFX extraction column and leave for 1min. Spin for 1.0 min at 10000g in
the Jouan 4i. Wash the extraction column with 0.5ml of wash buffer by spinning
through for 1.0 min at 10000g in the Jouan 4i. Place GFX extraction column
in fresh microtube and add 50µ of water. Leave for 1min and spin
for 1.5min as before.
Add 5µl of 3M sodium acetate, pH7.0 and 150µl of ethanol to
the eluate. Mix and spin at full speed for 15min to precipitate the purified
DNA fragment. Wash with 200µl of 70% ethanol and allow to dry on
the bench for 10 min.
Add to the dry pellet 0.5-1.0µl (10-20ng) of suitably cut plasmid
vector, 1µl of 10X ligation buffer (Helena), water to 9.5µl
and 0.5µl of high concentration T4 DNA ligase (Helena). Mix and leave
at room temperature for 4 to 16 hours.
Proceed to copmpetent cell transformation and plating
Obviously gel purified inserts will result in plasmids bearing the
required product. However sometimes it is desirable to ligate a mixture
of restriction products, such as those from a recombinant l
phage DNA digest, resulting in plasmids bearing a variety of restriction
products. It is simple to digest such target materail with enzyme(s) of
choice and then extract and precipitate the reaction mixture (as with the
production of cut plasmids) without any gel purification step, and then
use directly in ligation reaction as described above.
Plasmids and digests may also be double digested or sequentially digested
and filled-in after digestion to generate partially filled or blunt ends.
LB media; 10g tryptone, 5g yeast extract, 5g NaCl
LB agar; LB media with 10g/l agar
50X TAE buffer; 2M TRIS/Acetic acid, pH 8.0, 250mM sodium acetate, 50mM