Transgene Preparation – BAC DNA Protocol

The GEM Core can successfully generate transgenic mice with transgenes derived from BAC's. The efficiency of generating transgenic mice with BAC DNA is less than that of traditional transgene constructions. As with traditional transgene constructions, successful generation of BAC transgenics is highly dependent upon the preparation of the transgene DNA. If the transgene is improperly purified, the usual result is that embryos will survive microinjection but the recipient moms will not get pregnant.

The investigator is responsible for preparing the BAC transgene DNA for injection. The BAC injection is scheduled in advance through iLab and the BAC DNA is delivered to the GEM Core, N630. The sample must be attached to a copy of the request. Below is a protocol for the preparation of BAC DNA for microinjection. The protocol was prepared by Eric Nemoz-Gaillard a postdoctoral fellow in the laboratory of Dr. Ming Jer Tsai and Xuan Chi a graduate student in Dr. Robert Schwartz's Laboratory. Any questions concerning BAC DNA isolation direct to Memsmail@bcm.edu

Protocol Modified From:

Human Genome Sequencing Center [BCM] [steps 2-19]

Welcome Trust Centre for Human Genetics and Institute of Molecular Medicine, Oxford [HGIMM] [steps 20-23]

University of Michigan - Transgenic Animal Model Core
[U. Mich.] [alkaline lysis 2-11 & step 27]

The Nest Group [Nest Grp] [step 27]

Purification of BAC DNA for Microinjection

1. Make a schedule with GEM Core: This protocol will take 5 days. Therefore, purify the DNA 6-7 days before your scheduled microinjection date, to minimize degradation and allow one day for running 1ul of purified BAC DNA on a pulse field gel.

2. Inoculate 800mL (2x400ml) of LB medium containing relevant antibiotics with 0.5 ml of an overnight culture. It is very important to start from a single freshly streaked colony. Shake at 37ºC overnight (full 16-18 h. No longer than 18h).

3. Pellet the cells at 2,000g (4,000rpm for a JA10 rotor) for 15 min.

4. Discard supernatant, making sure to drain fully.

5. Resuspend each pellet completely with 30ml/400ml starting culture of chilled buffer P1 (no RNase).

6. Add 100,000U Ready-Lyse (Epicentre) to each 400mL starting volume, incubate at RT for 15min.

7. Carefully add 30mL of buffer P2 (by washing the side of the bottle with P2) and incubate at RT for 5min. DO NOT MIX OR VORTEX. If cell lysis is not well achieved, roll the tubes on bench top very gently.

8. Again, carefully add 30 ml of chilled buffer P3 (by washing the side of the bottle with P3) and incubate on ice for 15min, DO NOT MIX OR VORTEX.

9. Spin at 15,000g (9000rpm for a JA10 rotor) for 30 min at 4ºC.

10. Gently pour the supernatant into a clean bottle and repeat the spin for an additional 15 min at 15,000g (9000rpm for a JA10 rotor).

11. Transfer the supernatant through Clontech's filter paper to a third bottle and add 45 ml (half of the added volume of P1+P2+P3), mix gently, but thoroughly.

12. Spin at 6,000g (7,500rpm for a JA10 rotor) for 15min at 4ºC.

13. Decant the supernatant and drain the pellet completely.

14. Dissolve the pellet with 9mL standard TE and transfer to 30mL high-speed centrifugation tubes.

15. Add 4.5ml of 7.5M K-Acetate (autoclaved), mix very gently and place at -80ºC for 30min.

16. Completely thaw into a RT to 30ºC water bath and centrifuge at 5,000g (6500rpm for a JA10 rotor) for 12min.

17. Pour the supernatant into another 30ml centrifugation tube and repeat previous centrifugation step.

18. Mix with 27ml of 100% ethanol and spin at 5,000g (6500rpm for a JA10 rotor) for 12min.

19. Resuspend the pellet with 1mL of buffer P1 or S1(with RNase), incubate at 37ºC for 30min.

20. Add 100uL of 10% SDS and mix. Add proteinase K to a final concentration of 0.2mg/mL. Digest at 50-60ºC for 3h or overnight.

21. Extract the DNA solution with Buffered phenol (pH8). Do not VORTEX. (Phase Lock Gel Heavy (Eppendorf) can help the extraction steps).

22. Extract the DNA solution with Chloroform, successively. Do not VORTEX. (Phase Lock Gel Heavy (Eppendorf) can help the extraction steps).

23. Extract the DNA solution with Chloroform, successively. Do not VORTEX. (Phase Lock Gel Heavy (Eppendorf) can help the extraction steps).

24. Add 7.5 M Ammonium Acetate for a final concentration of 2.5M (0.5vol). Precipitate DNA with 0.5vol isopropanol at room temperature, mix thoroughly.

25. Centrifuge in 1.5mL Eppendorf tubes at 12,000rpm for 5min.

26. Wash pellet with 1ml 70% EtOH, add 300uL of 50:10 TE buffer to the pellet ( to improve DNA stability), incubate at 37ºC for 30 min and leave at RT overnight or for at least 3 hr.

27. Add 12ml of Clontech's buffer N2 (Equilibration buffer) and proceed through pre-equilibrated columns as prescribed by the manufacturer. Resuspend in TE 50:10. Do not let the DNA dry too long (translucent, not white), incubate at 37 ºC for 30 min and let resuspend overnight.

28. Linearization: Digest with terminase (or other appropriate enzyme), for at least 3h to overnight.

29. Phenol:chloroform DNA purification. Let the pellet dry for 15 min and dissolve in 20ul of newly filtered BAC microinjection buffer for 30 min at 37 ºC.

30. Quantification:

a. OD reading: Microinjection DNA must meet the following parameters: 260/280 of 1.70 to 2.0, (<1.7 indicates protein contamination, >2.0 indicates phenol and/or Chloroform contamination. In both cases, you need to ethanol precipitate DNA one more time).

b. Digest with frequent cutter, like BamHI, and run on a 0.8% agarose gel, you should see a ladder pattern with sharp bands and no smear.

c. Run 1uL of linearized DNA on a pulse field gel. You should see a sharp band of expected size, no smear nor genomic DNA contamination.

31. DNA storage: Store resuspended DNA at 4ºC until microinjection day.

32. On the morning of microinjection, thaw DNA, do OD reading again, dilute into 1ng/ul in BAC microinjection buffer and send it to microinjection core on ice. Higher concentration might be toxic.

General Buffers

P1 (50:10TE): 50mM Tris-HCl, 10mM EDTA, pH 8.0. Store at 4ºC
P2: 200mM NaOH, 1% SDS. RT
P3: 2.8M K-Acetate, pH 5.5. Store at 4ºC
7.5M K-Acetate: 184g K-Acetate in 250mL sterile water. Filter sterilize or autoclave

BAC DNA Microinjection Buffers

(Adapted from University of Michigan, Transgenic Animal Model Core , This site also contains other protocols for preparation of BAC DNA for microinjection.)
10 mM tris, pH 7.5,
0.1 mM EDTA,
30 µM spermine,
70 µM spermidine,
100 mM NaCl.

Stocks

1000x Polyamine Stock
30 mM Spermine (Sigma, tetrahydrochloride, #S-1141)
70 mM Spermidine (Sigma, trihydrocholoride, #S-2501)
Dissolve the spermine and spermidine together in autoclaved distilled water, filter sterilize (0.2 micron filters), and store at -20ºC. Since the polyamines are very hygroscopic, it is suggested that small quantities (1 gram) should be ordered and then all of it should be prepared at once.
1M Tris-HCl, pH 7.5 (autoclaved)
0.5 M EDTA, pH 8.0 (autoclaved)
5 M NaCl (autoclaved)

Stocks

Stock

Final Concentration

Volume

1M Tris-HCl, pH 7.5)

10 mM

0.5 ml

0.5 M EDTA, pH 8.0

0.1 mM

10 microliters

5 M NaCl

100 mM

1 ml

1000x Polyamines

1x

50 microliters

H2O


Bring to 50 mls

Prepare fresh and discard unused microinjection buffer.

Schedl A, Larin Z, Montoliu L, Thies E, Kelsey G, Lehrach H, Schutz G. 1993. A method for the generation of YAC transgenic mice by pronuclear microinjection. Nucleic Acids Res 21:4783-7.

Montoliu L, Bock CT, Schutz G, Zentgraf H. 1995. Visualization of large DNA molecules by electron microscopy with polyamines: application to the analysis of yeast endogenous and artificial chromosomes. J Mol Biol 246(4):486-92.

Regents

Regents

Company

Catalog #

Amount

Price

Ready-Lyse

Epicentre

R1082M

2x106U

$80

Ready-Lyse

Epicentre

R1082M

4x106U

$140

Proteinase K

Boehringer Mannheim

1 964 364

1.25mL

$53

Proteinase K

Boehringer Mannheim

1 964 372

5.0mL

$111

Proteinase K

Boehringer Mannheim

1 964 399

25mL

$475

Clontech BAC Maxi Kit

Clontech

K3008-1

10 BAC Maxiprep

$175

NucleoBond AX500

Clontech

4003-1

10 columns

$125

Columns

Clontech

4003-2

50 columns

$621

Buffer set I

Clontech

4040-1

For 10 classic Maxiprep

$40

General Consideration for BAC Purification and Handling:

1. It is advised to maximize precautions during BAC DNA handling. For this reason do not vortex, pipet up and down or repeatedly freeze-thaw. Use wide bore tips if repeated manipulations of the DNA are necessary. It is recommended to store the BAC DNA at 4 ºC (several weeks) or to freeze aliquots at -80ºC.

2. Concentration measurements by UV spectrophotometer are not very reliable since you can have absorbing contaminants, as well as, significant amounts of E. coli DNA. Therefore, pulse field gel electrophoresis of Not I digested BAC DNA is the method of choice to evaluate the integrity, quality and quantity of BAC DNA.