Cloning Partial Digests
April 22, 1998 ECK
A guaranteed but inefficient way to clone a selectable marker is to select it from a pool of cloned partially digested chromosomal DNA. Sau3A is the most commonly used nuclease. It cuts about every 256 bp, has a relatively unbiased nucleotide composition, is insensitive to dam methylation, and generates a 5' overhang compatible with BamHI, BclI and BglII overhangs. Other enzymes occasionally used are TaqI, AluI and DNAse A followed by blunting with T4 DNA Polymerase.
1. Partial Sau3A Digest (Based on the protocol described in Sambrook et al, 9.24-9.28, 1989)
Begin with 200 µL of a standard chromosomal DNA preparation (>0.1 µg/µL).
Label 10 Eppindorf vials 1 - 10.
Add to the DNA
Dispense 40 µL to tube 1, and 20 µL to tubes 2 - 10.
- 20 µL 10X Sau3A Buffer (New England Biolabs)
- 2 µL BSA at 10 mg/mL
From this point until incubation, keep everything on ice!
Add 1 µL of Sau3A at 10 units/µL (New England Biolabs) to tube 1.
Transfer 20 µL to tube 2.
Mix, transfer 20 µL to the next tube
Repeat the last 2 steps up to tube 9. Do not transfer to tube 10, which will serve as a no-digest control.
Incubate all 10 tubes 1 hr at 37°C.
Kill the digest 20' at 65°C.
Prepare a 0.8% agarose gel with at least 12 lanes.
Load a size standard in first lane 1, skip next lane and load the remaining lanes with digests 1 - 10.
Run the gel and stain with ethidium bromide in the usual fashion.
Calculate the minimum size digest product which will contain the marker. For instance, a Tn10dCam insert contains a 660 bp chloramphenicol resistance gene. Add a minimum amount necessary to give enough adjacent sequence to fulfill the design of the experiment. In the case of Tn10dCam, you would probably want at least 200 bp of adjacent DNA for sequence analysis or 1 Kb for genetic characterization. The resulting number is the lower size limit needed. Add to this 2 to 3 Kb to get the upper size limit.
Inspect the gel for lanes containing digest smears which are most intense between these to limits. Excise a rectangle of gel between these two limits and including the indicated lanes.
Purify the DNA using a convenient methodology. Qiaquick works well for me. The final volume should be about 100 µL. DNA concentration is impossible to predict.
3. Vector Digest and Dephosphorylation
Nearly all cloning vectors have unique BamHI or BglII sites in their polylinkers. Cut 5 to 10 µg of vector with one of these two enzymes in a 20 µL reaction using standard protocols.
Add 1 µL of Calf Intestinal Alkaline Phosphatase ("CIP") diluted to 5 units/µL in 1X buffer used in the restriction digest.
Incubate 1 hr at 37°C.
Add 1 µL EDTA at 100 mM and heat 10' at 75¡, to kill most of the CIP.
Phenol extract, followed by 2 butanol extractions.
Add 1 µL glycogen (Boehringer Mannheim, molecular biology grade), one third volume 7.5 M ammonium acetate, and 2.5 volumes 95% ethanol.
After 10' on ice, spin 10' at full speed in a micocentrifuge and discard the supernatant.
Wash pellet once with 70% ethanol, and dry slightly for about 5' under vacuum. The pellet will redissolve more easily if not allowed to dry completely.
Dissolve in 40 µL H2O.
Set up 9 20 µL ligation reactions, each containing 2 µL 10X Ligation Buffer (New England Biolabs, already contains ATP), 1 µL T4 DNA Ligase at 200 units/µL (New England Biolabs) and all 9 combinations of zero, 3 and 8.5 µL partially digested chromosomal DNA with 1, 3 and 8.5 µL cut vector from the previous step. Make up the balance with H2O.
Incubate overnight at 16°C.
Phenol extract once, butanol extract twice and ethanol precipitate as previously described.
Redissolve in 10 µL H2O.
Electroporate into DH5a (E.coli) or TR6579 (Salmonella). These are restriction-minus methylation-plus strains which are easily transformed by electroporation.
Plate to selective medium and proceed per normal.
Last Update: Thursday, 19-Jun-2014 11:50:27 PDT
This page has been viewed 493 times.
eckofoid at ucdavis.edu