Subtrmethods

On this page you will find information regarding the BAC vector for library preparation, protocols for screening BAC libraries, BAC minipreps and BAC recombineering.

BAC Vector for Macro-Array Genomic Libraries

Frengen E., Weichenhan D., Zhao B., Osoegawa K., van Geel M., de Jong P. J. 1999. A modular, positive selection bacterial artificial chromosome vector with multiple cloning sites. Genomics. 58(3):250-3.

Abstract

To construct large-insert libraries for the sequencing, mapping, and functional studies of complex genomes, we have constructed a new modular bacterial artificial chromosome (BAC) vector, pBACe3.6 (GenBank Accession No. U80929). This vector contains multiple cloning sites located within the sacB gene, allowing positive selection for recombinant clones on sucrose-containing medium. A recognition site for the PI-SceI nuclease has also been included, which permits linearization of recombinant DNA irrespective of the characteristics of the insert sequences. An attTn7 sequence present in pBACe3.6 permits retrofitting of BAC clones by Tn7-mediated insertion of desirable sequence elements into the vector portion. The ability to retrofit BAC clones will be useful for functional analysis of genes carried on the cloned inserts. The pBACe3.6 vector has been used for the construction of many genomic libraries currently serving as resources for large-scale mapping and sequencing.

NB: pBACe3.6 clones have chloramphenicol antibiotic resistance. Clones should be grown in LB containing 12.5 ug chloramphenicol/ml. Further information on this vector is available from CHORI, Children's Hospital Oakland Research Center

BAC Library Screening

Materials:

Hybridization solution:

• 5x SSPE

• 0.1% NaPPi

• 5% (w/v) SDS

Stripping buffer:

• 0.1x SSC

• 0.1% SDS (w/v)

• 0.2 M Tris-HCl, pH 7.5

DNA Probes:

• See Agilent Prime-II Random Primer Labeling Kit

• Sephadex G50

Procedure:

Making the probe:

1. 25ng DNA template - ensure has no vector seq (included T3 etc site as this will cross react with the BAC backbone seq)

2. Add appropriate ul of H20 to bring 25 ng DNA to 23 ul total

3. Add 10 ul of random primers (total is now 35 ul)

4. Heat denature - boil 5 min

5. Remove to room temperature

6. Add 5 ul of 5X of dATP buffer

7. Add alpha ³²P dATP at 3000 Ci/mmol.

8. Add 1ul Klenow

9. Incubate at 37^°C for 10 min

10. Add 2 ul of stop mix

11. Take 1 ul probe mix and add to 99 ul 0.2 M EDTA mix

12. Run through Sephadex G50

13. Take 1 ul post spin and add to 99 ul 0.2 M EDTA mix

14. Spot 1 ul of pre and post spin onto Whatman filters

15. Use scintillation counter to measure specific activity

Screening Filters:

[CRITICAL] If using a filter for the first time, follow the stripping protocol before hybridization (see below)

1. Place the membrane(s) in glass bottles that fit in the Hybaid hybridization oven, using nitex sheets to separate the filters. A complete set of library filters will fit in one bottle for both hybridization and washing.

2. Prehybridize in a shaking water bath at 65°C (55°C for cross species probes) for 1 hr.

3. Remove HS to minimum amount - so that it just covers the filters Xul probe to the prehybridization solution.

4. Incubate for at least 12 hr at 65°C (55°C for cross species probes).

5. Following hybridization, wash the filters by incubating them in 2x SSPE, 0.1% (w/v) SDS at room temperature for 10 min. Repeat.

6. Replace the solution with 2x SSPE, 0.1% (w/v) SDS. Incubate at 65°C (55°C for cross species probes) for 15 min. Repeat.

7. Replace the solution with 1x SSPE, 0.1% (w/v) SDS. Incubate at 65°C (55°C for cross species probes) for 10 min. Repeat.

8. Replace the solution with 0.1x SSPE, 0.1% (w/v) SDS. Incubate at 65°C (55°C for cross species probes) for 10 min. Repeat. (Use this for high stringency).

9. Remove filter, wrap in plastic wrap and carry out autoradiography.

10. Plastic wrap should be employed without trapped air for best exposures. From the final wash, pick up the filter by one corner and allow it to drip dry for 10 seconds. Place the filter face up on a piece of plastic wrap still attached to the roll. Fold the attached edge off the filter, then roll the top layer of wrap onto the filter. This expels any trapped air Most importantly, do not allow the filter to dry until it is stripped.

Stripping filters:

Bring 0.5% SDS to a boil.

Pour on the membrane and allow to cool to room temperature.

(If necessary, repeat)

Storing filters:

Short-term storage (1 or 2 weeks)

a. Wet two sheets of Whatmann paper in EDTA-containing stripping buffer.

b. Place the membrane between the two wet papers.

c. Wrap the papers and the membrane with plastic wrap and keep them in refrigerator until reuse.

Long-term storage

a. Sandwich between two sheets of plastic wrap.

b. Expose the membrane to X-ray film for at least 12 hr to check whether stripping is done completely. If stripping is done completely, place the membrane between two sheets of dry Whatmann paper and dry it at room temperature for at least 24 hr (until completely dry).

c. If stripping is not complete, repeat steps 1&2 but execute step 2 at the higher temperature.

Determining microwell plate coordinates from arrayed filters:

The high-density filter array is a square arrangement of 48X48 blocks which can be thought of as six sub-fields of 16X24 blocks. Thus each sub-field is equivalent to the wells of a 384-well plate. Each block is a 4X4 array of eight clones spotted in duplicate. That is, the inoculum from each well of each plate has been spotted twice onto the filter in the same 4X4 block. The arrangement has been designed so that the two spots define a unique angle different from all the others within the 4X4 block. The unique angular relationship of the spot pair defines the plate from which that clone was taken. In the accompanying figure, a 4X4 block adjacent to each sub-field indicates the plate number assignments for the blocks in that sub-field. The position of a 4X4 block containing a positive spot pair can be described by the X-Y coordinates of the block in the sub-field (X coordinates are A through P, from bottom to top; Y coordinates are 1 through 24, from right to left). For filters beyond the first one (A) in the set, the plate numbers are increased in increments of 48, thus the plate number for the B filter is the decoded number plus 48; for the C filter, plus 96; etc.

EXAMPLE: On the accompanying figure there is a positive spot pair circled in white. It lies in the left-middle sub-field at X-Y position I-10. That is I blocks (9) up from the bottom of the sub-field and 10 blocks over from the centerline. Thus the well position with in the plate is I-10. For this sub-field, the angle of the spot pair within the block indicates #31. There fore, the clone is located on plate #31 in well I-10.

NOTES: Identification of spot coordinates in the case where the background on the filter is very low is aided by pre-marking the filters when they are dry and the colony residue is visible. Dry filters are marked by indentations from a ball point pen. The pen tip is pressed into a filter that is placed on a piece of 3MM paper on a hard surface while observing the operation in oblique lighting. Dots can thus be made at the boundaries of the 6 sub-fields and at the extreme corners of the array.

To aid orientation in the newer filter sets, the A1 well for each plate has been left empty. Thus there are six empty squares that have no bacteria on the filter. After hybridization these squares have lower background and will orient the filter. The squares are 3 sets of 2 across when the label is on the upper right hand edge.

BAC Miniprep Protocol

This protocol uses alkaline lysis and precipitation to isolate BAC DNA to analyze by Pulsed-field Gel Electrophoresis, PFGE, or PCR. BACs purified using this protocol cannot be injected into fertilized eggs.

Materials:

Buffer P1: Stored at 4^oC. Add the RNAseA just prior to use.

• 15 mM Tris, pH 8.0

• 10 mM EDTA, pH 8.0

• 100 μg/ml RNase A

Buffer P2: Make fresh each use.

• 0.2N NaOH

• 1% SDS

Buffer P3: Cool on ice prior to use.

• 3M KAc pH 5.5

Procedure:

1. Inoculate a single bacterial colony into 3 ml LB containing 12.5μg/ml chloramphenicol in a 14 ml culture tube. Grow overnight (< 16 hrs), shaking at 250-300 rpm.

Optional: make bacterial glycerol stock (15%) of BAC.

2. Pellet the bacteria by transferring 1.5 ml of each culture to a 1.7 ml microcentrifuge tube and centrifuge at 6800 g for 3 min. Discard supernatant.

3. Repeat step 2.

4. Resuspend each pellet in 250 μl P1 carefully. Be sure to fully resuspend until suspension is creamy with no clumps.

5. Add 250 μl P2 and invert tubes 5 times to mix. The appearance of the suspension should change from very turbid to almost translucent.

6. Add 350 μl cold P3 slowly to each tube and shake gently during addition. A thick white precipitate consisting of E. coli DNA and protein will form. Invert the tube several times to mix the solution thoroughly.

7. Place the tubes on ice for 5 min.

8. Centrifuge at 18,000 x g for 10 min at room temperature to pellet the white precipitate.

9. Transfer the clear supernatant (~700-800 μl) to a 1.7 ml microcentrifuge tube.

10. Spin again in a microcentrifuge for 5 min at RT to remove the rest of the debris. Transfer the clear supernatant to a fresh tube.

11. Add 0.8 ml ice-cold isopropanol. Mix well by inverting tubes ~10 times. Place the tube on ice for 30 min, or leave overnight at 4°C.

12. Centrifuge at 18,000 x g for 30 min at 4^oC to pellet BAC DNA.

13. Remove supernatant and add 1ml of ice-cold 70% EtOH. Invert tubes several times to wash the DNA pellets. Centrifuge at 18,000 x g for 15 min at 4^oC.

14. Repeat step 13.

15. Centrifuge at 18,000 x g for 2 min at 4^oC to remove residual EtOH. Carefully remove all supernatant, taking care not to dislodge the pellet.

16. Briefly air-dry pellet at room temperature.

17. Resuspend pellet in 20-30 μl TE (10 mM Tris; 1 mM EDTA). Gently flick the bottom of the tubes to resuspend DNA. Do not vortex or pipet up and down.

For storing use high EDTA TE - i.e. 10mM Tris 10mM EDTA.

To analyze the BACs, use 6 μL of this prep in a NotI digest to run on a PFGE.

For PCR dilute 1 μl of this prep in 24 μl TE.

BAC Recombineering

More information is available here: Techniques for analyzing gene expression using BAC-based reporter constructs. Buckley KM, Ettensohn CA. Methods Cell Biol. 2019;151:197-218. doi: 10.1016/bs.mcb.2019.01.004. Epub 2019 Feb 23. PMID: 30948008 Review.

Materials:

Reagents:

LB with kanamycin (25 μg/mL)

LB with chloramphenicol (12.5 μg/mL)

SOC media

10% L-(+)-arabinose

Gel extraction kit

High fidelity DNA polymerase

DpnI

3 M NaOAc (pH 5.2)

Cell lines:

1. Electrocompetent DH10B

2. EL250. A DH10B-derived strain that contains a λ prophage with the recombination genes exo, bet, and gam. These genes are repressed by the temperature-sensitive repressor cI857.

Procedure:

Prepare the recombination cassette

Recommendations for designing the “recombination arms” are available in Buckley KM, Ettensohn, CA

Amplify the recombination cassette (RC)

1. Use high fidelity DNA polymerase to amplify the RC with both arms (amplify from the 5′ end of the 5′ arm to the 3′ end of the 3′ arm) from plasmid DNA. Minimize the amount of plasmid DNA used in PCR.

2. Run the product on an 0.8% agarose gel and purify the fragment using a commercial gel extraction kit.

3. Treat the amplified RC with 5 U DpnI in the appropriate buffer.

4. Incubate the reaction at 37^oC for 1 hour.

5. Heat the reaction at 65^oC for 15 minutes.

6. Precipitate the recombination cassette by adding 0.1 volume sodium acetate (3 M, pH 5.2) and 2 volumes cold ethanol (100%).

7. Incubate at -20^oC at least two hours to overnight.

8. Pellet the DNA by centrifugation (maximum speed, 4^oC, 30 minutes).

9. Wash the pellet with 70% ethanol, dry briefly and resuspend in 20 μl H 2 O.

Transform the BAC into EL250 cells

1. Pick a starter culture of EL250 cells from frozen stocks or fresh streak in 3mL LB (no antibiotic). Incubate shaking overnight at 30-32°C.

2. Dilute the culture 0.7 mL in 50 mL fresh LB. Incubate for ~5 hrs at 30°C, until O.D.600 is 0.8-1.0. From this point on, KEEP ON ICE AT ALL TIMES.

3. Centrifuge bacteria for 10 min, 4°C, 3000 rpm using a pre-chilled 50 mL conical.

4. Resuspend in 50 mL ice-cold dH2O and spin down as above.

Tip: resuspend the pellet first in 1 mL, but avoid pipetting. Instead rock/shake the tube briskly against and within the ice bucket. This takes time, but is necessary to better preserve the cells. Once the pellet is resuspended in a small volume, top off to 50 mL and mix gently by inverting a few times.

5. Repeat steps 3 – 4.

6. Centrifuge bacteria for 10 min, 4°C, 3000 rpm. Resuspend in 1 mL ice-cold dH2O as above. Transfer to chilled 1.5 mL tube.

7. Centrifuge for 2 min, 4°C, maximum speed.

8. Wash 3 times with ice-cold dH2O.

9. Use the cells immediately for electroporation. Washed, electrocompetent cells can be frozen @ -80°C with 10% f.c. glycerin, but transforming efficiency will decrease. It is best to use fresh cells every time.

10. Add 200 ng of BAC DNA (~1/2-1/3 of a mini-prep) to 10 μl electrocompetent EL250 cells + 10 μl dH2O on ice. Transfer to ice-cold 0.1 mm electroporation cuvette.

11. Electroporate at 1.4-1.7 kV/cm, immediately add 1 mL S.O.C. or LB to the cuvette, and transfer to a 15 mL culture tube. BAC transformation work better at slightly lower voltages (1.4-1.7 kV/cm). The bigger the BAC, the lower the kV/cm.

12. Incubate shaking at 30°C for 90 min.

13. Plate everything on LB/chl plates and incubate up to 24 hrs at 30°C. Using the large plates increases quality of colonies and overall efficiency.

14, Pick a few colonies and grow at 30°C overnight to 24 hr in LB/chl.

15. To verify that the transformation worked correctly, mini-prep the BACs, digest with NotI and analyze on a PFGE.

16. Make glycerol stocks of positive EL250-BAC clones.

Recombineer the fluorescent protein into the BAC DNA

1. Pick a starter culture of EL250-BAC cells from frozen stocks or fresh streak in 3 mL LB/chl and incubate overnight at 30°C.

2. Dilute the culture 1 mL in 50 mL fresh LB/chl and incubate for ~6 hrs at 30°C, until O.D.600 is 0.8-1.0.

3. Place in pre-warmed 42°C shaking water bath for 15 min to activate the recombinase genes.

If a shaking water bath is not available, gently swirl the flasks by hand for the entire 15 min, taking care to keep the bottom fully submerged in the bath. Due to this step (and the washing steps thereafter), it is difficult to prepare more than two BACs simultaneously.

4. Immediately chill by swirling in ice-water slurry for ~10 min.

5. Transfer to a pre-chilled 50 mL conical tube, pellet and wash as described above (on ice).

6. Add 200 ng of FP cassette DNA to 10 μl electrocompetent EL250-BAC cells + 10 μl dH2O on ice.

7. Transfer to ice-cold 0.1 mm electroporation cuvette. Electroporate at 1.8 kV/cm and immediately transfer to 1 mL S.O.C or LB. Incubate at 30°C for 1 hr. Plate 50 μl and 500 μl on LB chl/kan plates and incubate 24 hrs at 30°C.

8. Colonies picked right after overnight incubation @ 30°C have free FP cassette in them, whereas those picked after a full 24 hr of incubation have recombined FP into the BAC. An EL250 cell with recombined BAC may grow slower due to low copy number of the BAC vector.

9. Check the recombination by restriction digest or PCR.

10. Make glycerol stocks (15%) of EL250-BAC/FP clones.

Remove the kanamycin cassette

1. Start an overnight culture of EL250-BAC/FP cells in 3 mL LB chl/kan. Dilute culture 1:50 in 25 mL of LB/chl. Grow at 30°C for ~5 hrs until O.D.600 is ~0.5.

2. Add 0.25 mL 10% L-(+)-arabinose and grow for 1 hr at 30°C. **Note: D-(-)- arabinose will not work. It has been tried.

3. Dilute culture 1:10 in 10 mL of LB/chl and grow for 1 hr at at 30°C.

4. Streak 2 μl of this culture on an LB/chl plate. Grow overnight at 30°C.

5. Select colonies that have lost the kanamycin resistance cassette by streaking clones (~4) onto both LB/chl and LB/chl/kan plates and grow at 30°C overnight. The colonies that do not grow on the LB/chl/kan plates have successfully eliminated (flipped) the kanamycin resistance cassette. This step is very efficient and gives nearly 100% flipped clones.

6. PCR diagnostic and sequencing can be used to confirm removal of kan cassette.

7. Make glycerol stocks (15% glycerol) of EL250-BAC/FP/kan-flipped clones.

Transform the recombinant BAC into DH10B cells

1. Miniprep an overnight culture of EL250-BAC/FP/kan-flipped cells.

2. On ice, combine 1 µl of the BAC DNA (miniprep) with 10 µl commercial DH10B electrocompetent cells + 10 µl dH2O.

3. Transfer to ice-cold 0.1mm electroporation cuvette. Electroporate at 1.4 kV/cm and immediately transfer to 1 mL S.O.C or LB.

4.Incubate at 37°C for 1 hr. Plate 50 µl and 200 µl on each of two LB/chl plates and incubate overnight at 37°C.

5. Pick a few colonies in 3 mL LB/chl and grow overnight at 37°C.

6. Miniprep these clones to confirm the size of the BAC insert and the FP recombination. Compare with the wildtype and non-flipped FP BACs as control. Analyze by PFGE.

7. Make glycerol stocks (15% glycerol) of this construct.