BAC DNA Isolation using 96 deep well blocks, pipetting on the Hydra 96, and growth in the HiGro
Fares Z. Najar, Mandi Aycock, Doris M. Kupfer and B. A. Roe
11-25-03 and updated on 03-09-06 for pEpiFOS-5 induction
and on 12-12-07 to add the Beckman 96 deep well block number
A. Culture Growth:
B. DNA Isolation:
- For BAC isolation for shotgun library construction:
- Pick a smear of colonies and inoculate 3 ml TB medium containing the appropriate
- Grow the culture for 8 hours, 37 degrees C, shaking at 250 rpm in the
Floor Shaker to produce the "starter culture".
- Add 1.8 ml of TB medium to each well in the 96 deep well block [Beckman (cat#140504)].
- Dilute culture 100 fold with TB and innoculate 5 ul of the diluted
culture into each well using the Eppendorf repeater pipette.
- Grow the BAC clone in the Floor Shaker for 18 hours at 37 degrees C
shaking at 350 rpm.
- For BAC or Fosmid isolation for end sequencing:
- Using the Hydra96, inoculate 2 ul of the BACs or Fosmids from their glycerol
plate stocks into either a 96 or 384 well shallow plate.
- Incubate at 37 deg C overnight in a static growth chamber without shaking
to produce the "starter culture".
- Add 1.8 ml of TB medium to each well in the 96 deep well blocks using the
Eppendorf repeater pipette.
- Using the Hydra96, inoculate 2 ul from this "starter culture" (either 96
or 384 well shallow well plates) into 96 well deep well blocks containing
1.8 ml of TB medium in each well.
- Grow the BAC clone in the Floor Shaker or HiGro for 18 hours at 37 degrees C
shaking at 350 rpm.
- Add 18 ul of 10x induction solution for amplification of the pEpiFOS-5
and incubate for an additional 5 hours at 37 degrees C shaking at 350 rpm.
C. Shearing and cloning
- Centrifuge the blocks at 3000 rpm for 10 mins. Discard the supernatant.
- *DO NOT FREEZE* the cell
pellets but continue with the next steps in the isolation without pausing.
- Add 300 ul of TE-RNaseA/T1 (TE-RNaseA/T1 is: 10 mM Tris-HCl, pH 7.6; 1 mM EDTA; 42 ug/ul
RNase A; 0.04 U/ul RNase T1) to each well using the Hydra (The Hydra will add 300 ul by
adding 150 ul twice).
- Incubate on the table top microtiter shaker at speed of 7 until for 20 minutes to fully
resuspended the cells.
- Add 300 ul of lysis buffer (1% SDS, and 0.2 N NaOH) to each well, using the Hydra.
Incubate on microtiter shaker at speed of 4 until a clear lysate is developed, takes
approximately 15 minutes.
- Add 300 ul 3M NaOAc, pH 4.5 to each well using the Hydra. Shake in the HiGro at 37degC
and a shaker setting of 400 rpm for 30 minutes.
- Freeze the blocks at -70 degrees C overnight.
- Remove the blocks from freezer and thaw. Centrifuge at 3200 rpm for 45 mins in a Beckman
CS-6R table top microtiter plate centrifuge.
- Transfer 500 ul of supernatant from each well to new blocks using the Hydra (The Hydra
will transfer 500 ul by transferring 250 ul twice).
- Add 500 ul of isopropanol to each well using the manual Eppendorf repeater pipette.
- Centrifuge the blocks at 3000 rpm for 30 mins. in the Beckman CS-6R table top microtiter
plate centrifuge to collect DNA. Decant the supernatant. Invert on a paper towel.
- Dissolve the pellet by adding 40 ul of steril double distilled water
(instead of 10 ul of 10:1 TE to each well which should be added if the BAC or Fosmid
DNA is to be used for shotgun cloning) using the Hydra followed by
shaking on the table top for 20-30 minutes to dissolve DNA.
- For isolating BACs and Fosmids for end sequencing skip to the last step, i.e. electrophores an alaquot of the dissolved DNA on a 1% agarose gel
as the second acetate cleanup step is not necessary. Typically 10 ul of this 40 ul
DNA solution is used for each end sequencing reaction with 1:8 Big Dyes or Amersham ET
mixes, 2 ul of universal primer from a 6.5 umolar primer stock solution, in a final
volume of 14 ul followed by 60 cycles of cycle sequencing amplification and loading onto the ABI 3730xl.
- For BAC or Fosmid DNA isolation for shotgun sequencing:
- Pool the contents of each well into two eppindorf tubes (~500 ul in each tube)
- Add 250 ul of 7.5 M of KOAc** to each well, using the Eppendorf repeater pipette.
- Freeze the tubeds in -70 degrees C for at least two hours to overnight.
- Thaw. Centrifuge at 13000 rpm for 30 mins. in a microfuge.
- Transfer ~700 ul of the supernatant to TWO new eppindorf tubes (350 ul each). Now the
total number of eppindorf tubes is 4.
- Add 700 ul of 95% ethanol.
- Centrifuge at 13000 rpm for 30 mins. in a table top microfuge. Decant.
- Wash by adding 1 ml of 70% ethanol to each tube
- Centrifuge at 13000 rpm for 30 mins. in a table top microfuge. Decant
resuspend DNA in 200 ul of 10/0.1 Tris/EDTA pH8.0. The typical yield is ~130 ug from
one deep well block.
- Run 1% agarose gel for qualitative assessment.
- Shear 50-100 ug DNA with the Hydroshear (Gene Machines, Inc)
set to generate 2-4 Kb size fragments.
- Precipitate the nebulized DNA using EtOH-OAc and wash with 70% EtOH
- Resuspend in 20 ul of dd-water.
- Fill-in and kinase treat the sheared DNA according to the standard protocol.
- Run a low-melt agarose gel for size selection.
- After excising the desired size range, extract DNA using the "freeze and squeeze"
method with 2 rounds of freezing or the ultrafree-DA centrifugal filters from
- Precipitate the DNA using EtOH-OAc and wash with 70% EtOH.
- Dissolve the DNA in 6 ul dd-water and use the entire solution for ligation and electroporation
into pUC18 using the standard protocol.
**7.5 M KOAc - 736.13 gm/liter final volume - Starting with a small volume of
ddwater (approximately150ml) slowly add the dry potassium
acetate and mix as it is added. Then add additional water and the remainder of the acetate
until all is dissolved and the final volume is 1 liter.
DO NOT ADJUST THE pH as the acetate will come out of solution.
Bruce Roe, firstname.lastname@example.org