Growing up of BAC clones

1. BACs are usually supplied as stabs in agar (2.5 ml screw-top tubes filled with LB agar, stabbed with a toothpick which has been put into a bacterial colony). These are then frozen; at -70℃ they will keep indefinitely, at -20℃ for a year or more, at 4℃ for several months and at room temperature (e.g. during shipping) for a week.

2. Make up 10 ml LB broth + 3 μl of chloramphenicol (dissolved in alcohol, 50 mg/ml, so final concentration is 15 ug/ml, stored at 4℃) for each stab which will be grown up.A

3. Dip sterile toothpick into stab, then drop whole toothpick into LB broth and put on cap.

4. Shake vigorously at 37℃ overnight.

Making single colony preps

1. Make LB agar plates with 2 ul/ml chloramphenicol (stock 50 mg/ml).

2. Dip inoculating loop into liquid culture from above. Make three parallel lines (without re-dipping) to streak bacteria out. Pass loop through flame, and make another three parallel lines at as shown, repeat twice more.

3. Place plates at 37℃ overnight to grow.

4. Ideal appearance is continuous line of colonies in lines 1, going to individual colonies clearly separated in lines 4.

5. Pick individual colonies for isolation of BACs by pushing a sterile toothpick into the colony.

Small-scale isolation of BAC DNA from 10 ml cultures .

Based on protocol from Zhang from Texas A&M University - http://hbz.tamu.edu/manual.pdf

Particular points to note are gentle shaking and inversion during the DNA isolation: these lead to differential precipitation of unwanted bacterial genomic DNA (4.6 MegaBases) and the BAC DNA (40 to 200 kiloBases) and keep the DNA in large molecules.

1. Stab a single colony from the agar plate above with a sterile toothpick and drop into 10 ml LB medium (or 20 ml, Abbott) with 25 μl chloramphenicol solution (50 mg/ml) in a 50ml tube. (This is 10X higher than ofen used in Abbott's lab.)

(A fast and dirty method is to use 10μl of a liquid culture for the inoculation but this is not recommended as it is important that a colony from a single cell is grown up to ensure purity)

2. Grow overnight with rapid shaking at 37℃ - 24 hr is good for the culture period.

3. Centrifuge culture at 1500g for 10 min, discard supernatant into waste container containing a few ml of bleach, and resuspend the pellet in the remaining 100 μl of culture medium. Add 100 μl of lysis buffer (50 mM glucose, 10 mM EDTA pH 8.0, 25 mM Tris.HCl pH8, autoclaved and stored at 4℃; Abbott lab: Add 4.5 μl of 10mg/ml RNase), shake gently and incubate on ice for 5 min. (Abbott lab: remove all culture medium that is possible and add 300 μl of GTE/RNase, resuspend by hard vortexing.)

4. Add 400 μl of 0.2 M NaOH, 1% SDS solution, shake gently and put on ice for 5 min (Abbott lab: 300 μl for a few seconds: as long as 5 min will denature BAC DNA irreversibly.).

5. Add 300 μl of 3M KOAc (potassium acetate adjusted to pH 5 with acetic acid) and incubate on ice for 15 min. (Abbott: not necessary to wait as long.)

6. Centrifuge at 2800g for 15 min. (Abbott: transfer to 2ml microcentrifuge tubes and spin 15000 g 15 min.)

7. Transfer supernatant (c. 750 ul) to microcentrifuge tube, add 450 μl isopropanol (Abbott: equal volume of isopropanol), mix and centrifuge at 12000g for 5 min.

8. Discard supernatant, wash pellet with 70% ethanol, centrifuge 12000g for 2 min.

9. Invert tube over a tissue and tip out ethanol and allow pellet to air dry for 20 min (less in dry atmosphere - it should not become fully dry)..

10. Resuspend pellet in 40 μl TE (pH 8) or water, allowing to redissolve at room temperature overnight or by heating up to 65℃. Do not vortex! (Resuspending in water means the DNA only lasts a few weeks at -20℃; using TE inhibits future restriction digestions; Abbott lab: suggests 0.1 mM Tris pH 8 for resuspension.)

11. Treat with RNase:: add 2μl of 10mg/ml DNase-free RNase and incubate for 30 min at 37℃.

12. Make a test digest of the BAC DNA:

10 μl of BAC DNA solution in TE/water

25.5 μl water (molecular biology grade, purchased from Sigma etc)

4 μl 10x restriction enzyme buffer

0.5 μl of restriction enzyme ( Hae III is often a good choice)

Digest overnight (min 3 hours) at 37℃ and run 10 μl in loading buffer on 0.8% agarose gel (0.5% can be better as it will separate fragments .5kb better), stain with ethidium bromide and photograph. Each lane of BAC DNA should have a number of discrete bands. It should digest almost completely (no DNA remaining in well), there should be no RNA (diffuse, large smear at bottom of gel) and each BAC should have a different and characteristic 'fingerprint' (pattern of bands).