John M. S. Bartlett.pdf - Bio-Nica.info

480 Horton, Raju, and Conti-Fine
1. Run a small portion of the reamplified product on a checking gel to make sure you have
enough of the correctly sized product with which to work. About 5 µL should give a clear
band. With care, you should be able to use this same gel to check your DNA at several
stages of the process. Do not let it dry out!
2. Add ~1 µg of uncut (supercoiled) plasmid vector to the tube containing the reamplified
material. This will both act as a carrier during the purification process and help to make
the effects of the restriction digests more obvious. You will probably not be able to see
the slight size differences resulting from cutting off the ends of the T-linkers, but you
should be able to see the difference as the vector is cut, and goes from being supercoiled
to being linear.
3. Extract the DNA twice with phenol:chloroform and once with chloroform.
4. Precipitate the DNA by adding 2 vol of 95% ethanol containing 2% potassium acetate to
the aqueous supernatant and spin on high speed in a microcentrifuge at 4°C for 30 min.
Discard the supernatant and carefully rinse the pellet with cold 75% ethanol.
5. Resuspend the DNA pellet in distilled water and add the appropriate 10× restriction
enzyme buffer. Add restriction enzymes and incubate until the vector is completely in the
linear form on a checking gel. Directional cloning will require cutting with two enzymes.
Manufacturers generally provide charts that indicate which buffer works reasonably well
with both enzymes. Run a lane with uncut vector on the checking gel. Uncut vector should
have three bands representing supercoiled, nicked circular, and (sometimes) linear forms.
Cut vector should only have the linear form. Even small amounts of uncut vector will lead
to high backgrounds on nonrecombinants.
6. Run the digested material on a preparative agarose gel and cut out the vector and insert
bands. Minimize exposure to ultraviolet light.
7. Recover the DNA separately from each band. Many protocols are available for this: We
usually use GeneClean for inserts larger than 250 bp.
8. Run about one-tenth to one-fifth of the DNA extracted from the band on a checking gel
to roughly estimate the amount of DNA recovered. The relative amounts of DNA in each
band are crudely estimated by visually comparing the brightness of the bands to those with
a known amount of DNA. The vector bands should contain more or less known quantities
of DNA, if you know how much you started with, and assume about 70% was recovered
from the preparative gel.
9. Mix vector and insert in at least a 21 molar ratio and ligate. Use about 100 ng of vector
per ligation. The ligation should resemble the following (for a larger insert, more DNA
is needed for the same molar ratio): 100 ng vector (2.5 kb), 20 ng insert (250 bp), 2 µL
of 10× ligation buffer, 1 µL of 10 mM ATP (if not in buffer), 0.5 µL of T4 DNA ligase,
and up to 16.5 µL of H 2 O.
10. Dilute the ligation 1:5, and use 1 µL to transform 20 µL of competent E. coli. Plate on
appropriate antibiotic medium.
11. Recombinant colonies can be screened using PCR. Depending on which restriction site
was used, you may be able to screen with a T-linker primer. For example, with inserts
cloned nondirectionally using the EcoRI site of the NdeT-linker, NdeTL-A can be used for
screening. Make a master mix for as many reactions as you need, in which a 10-µL reaction
contains: 1 µL of 10× PCR buffer, 1 µL of dNTPs (2 mM each), 1.5 µL of MgCl 2 (10 mM ),
2 µL of red sucrose dye, 1 µL of NdeTL-A (10 µM ), and 0.25 µL of Taq polymerase. Cover
these small reactions with oil while picking colonies to prevent evaporation.
12. Touch a recombinant colony with the tip of a sterile toothpick, dip the end of the toothpick
through the oil into the reaction, and swirl. Do not add enough bacteria to the reaction
to make it cloudy. Just a few bacteria are sufficient. Too much bacterial matter, or