1) Extract DNA from 40 to 50 flies with your favourite procedure. An easy method is described in the protocol for "Rapid small scale isolation of Drosophila DNA and RNA", p. 183 in this volume.
2) Check quality of DNA with desired restriction enzymes and Southern blotting. Usually 2 to 4 fly equivalents are enough to see a "good smear" on agarose gel. This step is optional, but K. (Mosey) Moses would not ommit it.
3) Digest 2 to 10 fly equivalents of DNA with an enzyme that fullfills desired criteria for rescue (If several different sites are suitable, try with more than one.) If possible use an enzyme that can be heat inactivated, otherwise include phenol/chloroform extraction before precipitation of DNA after digest.
4) Resuspend digested DNA in 20 to 50 µl TE. Use half of the DNA for the ligation. To avoid intermolecular ligation events, perform ligation reaction in a larger volume; 200 µl are usually fine. It is not necessary to add more than 2µl (e.g. 2 units of N.E. Biolabs enzyme) T4 DNA ligase. Incubate reaction mix for 4 hrs to overnight.
5) To 200 µl ligation mix add 20 µl of 3 M sodium acetate and precipitate with two volumes of ethanol. Spin, wash, dry and resuspend pellet in 10 to 20 µl TE. The DNA is now ready for transformation.
6) For bacterial transformation, I have used XL1-blue cells (Stratagene) and a high
efficiency transformation protocol as described by Hanahan (see below for details).
However, since more recently the electroporation procedure has been commonly used
and gives even higher transformation efficiency, this might be the method of choice
for plasmid rescue bacterial tranformation. When using electroporation, the cuvettes
should be sterile to avoid "surprises". Plate cells on Ampicillin or Kanamycin plates
depending on transformation vector used.
Comments:
The number of colonies per fly equivalent of genomic DNA varies greatly, ranging
from 0 to 30. The use of XL1-blue cells (10 to 100 times greater transformation efficiency
than other commonly used bacterial strains) and 25 C/ Hanahan transformation
protocol (see below) allows usually plasmid rescue from any transformant line.
Difficulties in obtaining clones from particular lines seems most likely due to an unusual
distribution of enzyme sites near the insert (try with different enzymes if possible).
When rescue of genomic upstream sequences is employed, the rescued plasmid
still contains the lacZ gene. For such plasmids, it is worthwhile to plate on plates
containg X-gal. Usually all XL1-blue colonies containing such plasmids will turn blue.
This might be usefull to destinguish the desired plasmids from potential contaminants.
Hanahan cells; high efficiency competent cells;
High efficiency (Hanahan) competent cells
1) Pick a single XL1-blue colony to 5 ml LB, grow to saturation at 37šC.
2) Inoculate 50 ml 2xYT + 10mM MgCl2 and 0.1 % glucose with 100ml of the seed culture in a
250 ml flask and grow at 25šC to A600 = 0.3 (about 12 hr).
3) Spin down cells in a 50 ml orange cap tube, resuspend cells in 20 ml ice cold TFB and
incubate on ice for 15 min.
4) Spin down cells and resuspend in 4 ml TFB.
5) Add 140 ml DnD and incubate on ice for 10 min.
6) Add 140 ml DnD, aliquot 200 ml per transformation and incubate on ice for 20 min.
7) Add up to 20 ml DNA and incubate on ice for 20 to 40 min.
8) Heat shock at 42šC for 90 seconds then return to ice for two min.
9) Add 800 ml 2xYT (+ 10mM MgCl2 and 0.1 % glucose), incubate at 37šC for one hour.
10) Plate, (if M13 phage wash the cells with two changes of medium first)
Solutions for Hanahan Cells:
TFB (for 500 ml) make 100 ml 0.5 M MES pH 6.3 FINAL
(9.76 g MES, pH with KOH, filter sterilise)
3.70 g Ultrapure KCl 100 mM
4.45 g MnCl.4H2O 45 mM
0.75 g CaCl2.2H2O 10 mM
0.40 g Hexamine CoCl3 3 mM
10 ml 0.5 M K-MES pH 6.3 10 mM
Distilled Water to 500 ml, filter sterilise, store at 4šC.
DnD 1.53 g DTT 1 M
9 ml DMSO 90 %(v/v)
100 ml 1 M Potassium Acetate (pH 7.5) 10 mM
aliquot and store at -70šC.
Reference: Hanahan, D., Techniques for transformation of E. coli
Ch 6 in DNA cloning, Vol I, A practical approach
Ed Glover, D.M., Oxford, IRL Press