Modified by Yue Hou March 2009
Large Scale Double-banded CsCl BAC prep, suitable for mouse embryo microinjection
Doug Mortlock, modified from (Kingsley Lab protocol/R. DiLeone/M.Koelle/R.Paro)
This protocol has been designed to minimize handling steps. It yields consistently good BAC
DNA for pronuclear injection. At all steps, avoid excess pipetting to minimize shearing of BAC
DNA. The CsCl gradient step is done twice to help remove sheared linear DNA. Doing it twice
substantially helps purify the intact circular BAC molecules, as we have found by pulsed-field
gel analysis of the BACs that show it reduces the background “smear” of sheared DNA. With
this protocol, we typically get transgenic founder rates of 10-15%.
Finally, I suggest always running 2 separate preps of the same BAC in parallel. Sometimes the
quality just varies from prep to prep. Run both samples on a pulsed-field gel when done, and use
the prep with the least visibly sheared DNA.
NOTES:
(DPM 2007) Mortlock lab: We are now using the MPB dept. 70i.Ti ultracentrifuge rotor. This
protocol has been updated accordingly. This gives better band separation than the vTi65.1 rotor.
(DPM 2006) •We are not currently using the polyamines step (appendix) much, as we have not
noticed an increase in efficiency with it. It is not strictly necessary (we have made many BAC
transgenics without it). However, some labs swear by it .
1. Day 1: Streak out BAC strain onto selective plate media to get isolated colonies.
2. Day 2: Inoculate single colony into 4 mls LB+antiobiotic miniculture, shake O/N (at least
24hurs).
3. Day 3: Use 2 ml of the miniculture to inoculate each 1L LB+antibiotic in a 4 L flask, shake
O/N. (Culture for minimum 16 hours if at 37˚ and 24 hours if at 32˚). So, you totally have two
4L flasks. Each flask contains 1L culture. Form now on, you treat these two flasks as separate
preps.
Day 4: Alkaline lysis
4. Spin down cells at 6K rpm, 10 min, 4˚ C, Sorvall F14s rotor or equivalent . Our centrifuge
bottle only can hold 250ml each time, so you spin twice to pellet 500ml culture in each bottle.
Then you should get 4 bottles of pellets for your total 2L culture.
You can stop here just by freezing your pellet bottles at –80˚ C till you are ready for the
following procedure.
5. Resuspend all the cells from 500 ml culture in cold 40 mls Solution I + RNAse A in one 250
ml centrifuge tube..
6. Add 40 mls fresh Solution II, mix gently for 30 sec. by gently inverting. Incubate at RT for 5
min. Cells should lyse completely by end of 5 min. The lysis should then be homogenous, with
minimal or no stringy opaque material.
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Modified by Yue Hou March 2009
7. Add 40 mls cold Solution III, invert several times to ensure it is mixed well (white ppt forms).
Incubate on ice 15 min.
8. Spin at 10K rpm, 20 min, 4˚ C.
9. To remove solid precipitated material, pour the supernantant filter paper(VWR, catlog#
28333-065) into a new 250 ml centrifuge bottle.
10. Add equal volume (120 mls) of R.T. molecular biology grade isopropanol, invert to mix, and
spin at 10K rpm, 20 min, 4˚ C.
11. Rinse pellet with 10 mls 70% ethanol, respin 10K rpm, 10 min., 4˚ C.
12. Carefully remove the 70% ethanol rinse and discard. Add 4.9ml TE pH7.4 into each bottle to
dissolve DNA pellet with gently rocking on nutator or rotary shaker. Here you have 4 bottles of
4.9ml solution.
13. Weigh out 9.8 g CsCl to a 15 ml tube. Transfer the 9.8ml DNA solution from step 12
(combine two bottles of 4.9ml solution together) into the tube and dissolve CsCl by rocking on
nutator or rocking platform at RT. When CsCl completely dissolves, add 0.98 ml EtBr solution
(10mg/ml) and mix. (Note for next step you will always need an even number of tubes so if you
have an odd number of BAC preps, prepare a balance CsCl tube now with TE+CsCl+EtBr).
14. Some solid material may precipitate out. Spin 15ml tubes at 3K rpm, 5 min. at RT to remove
ppt. material. Some of this may float at top, and can be removed by inserting a pipette tip into the
solution; most of it clings to the tip which is then discarded.
Avoiding precipitated material, transfer solution to new 15ml tube tube and respin.
Never keep the tubes at 4˚ C, which will prevent CsCl from dissolving into the solution.
Transfer final solution to ultracentrifuge tubes:
First, turn on the tube sealer and let it warm up ~ 10 minutes. Use Beckman Quick-Seal
polyallomer 13.5 ml tubes (Beckman cat# 342413), which will fit in the Beckman 70i.Ti rotor.
To load the DNA solution into the tubes, first insert a glass Pasteur pipette into the tube and then
add DNA solution to the pipette to let it fill up the tube. Fill the body of the tube completely and
then fill about 1/2 way up the narrow neck at the top the tube. Put a sealing cap on the top and
seal the tube (have someone show you how if you have not done this before). Inspect the tubes
to make sure they are sealed. Spin the tubes using 70i.Ti rotor at 18˚ C, 65K rpm for O/N (~16
hours).
15: “Pull” the band of BAC DNA:
How to do it: (Also see Sambrook, Molecular Cloning.) Remove tubes carefully from
ultracentrifuge rotor. In a darkroom, hold the tube with a clamp on a ring stand with a beaker
under the tube to catch any drops of solution. Gently insert 25g needle into top of tube at a
downward angle to provide a pressure outlet and leave it there. Set up a portable UV lamp so it
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Modified by Yue Hou March 2009
can illuminate the tube. Have a microcentrifuge tube ready. Turn out the white light, turn on the
UV and identify the DNA bands. The supercoiled BAC DNA that you want is the lower band of
the two bands that should be visible. Using a 1 ml syringe with 21 g needle , carefully puncture
the tube about 0.5 cm under the lower band. Syringe needles have a barb just behind the beveled
point, so the needle will break through suddenly after applying a bit more pressure – be careful!
Then, turn the beveled edge of the needle so the hole faces up against the lower BAC DNA band
and draw out the band by pulling on the syringe plunger. Extract the BAC DNA band in smallest
volume possible (should be < 1 ml). Do not over-pull too much – it is normal for the tube to
look like a little bit of the band got left behind after a “good” pull.
16. Transfer the DNA to a 15 ml tube. Bring volume up to 9.8ml by adding TE pH 7.4, and
repeat steps 13-15.
17. Butanol extraction: To remove the EtBr, extract with TE/NaCl-saturated butanol in 2 ml
eppendorf tubes, 6x times. To do the extractions, add an equal amount of TE/NaCl-saturated
butanol to the aqueous phase and mix well by inversion. (NEVER vortex BAC DNA!). The
EtBr will move to the upper butanol phase. Remove the butanol phase with a pipette tip and
discard as hazardous waste. Repeat 5 more times.
At this point, the salt still need to be removed and the BAC DNA must be equilibrated
into microinjection buffer. This is done in the next 2 steps. First we do cassette dialysis with
TE and then Centriprep column dialysis with microinjection buffer. The cassette dialysis is easy,
reduces number of washes in the column dialysis, and minimizes waste of microinjection buffer
(which is made with embryo culture grade water so is more expensive).
18. Slide dialysis: Prepare 1 L of TE in a beaker by combining 988 mls MilliQ-grade ddH20 +
10 mls Tris pH 7.4 + 2 mls 0.5 M EDTA pH 8.0 (autoclaved the solution with a stir bar inside
and cool to 4˚ C before use). For dialysis, use a Slide-a-Lyzer dialysis cassette (Pierce, cat
#66380, 10.000 MWCO, 0.5-3 ml capacity) and follow the manufacturer’s instructions. Hydrate
the cassette by immersing in the TE for at least 30 seconds. While it is hydrating, load the BAC
DNA into a syringe and attach a 21 gauge needle. Leave some air in the syringe behind the
DNA solution. Remove cassette and tap cassette edge on paper towel to remove excess liquid.
SLOWLY inject the BAC DNA into the cassette. Attach a float buoy (Pierce cat#66430) to the
cassette, and float the cassette in the TE beaker. Stir very gently in 4˚ cold room for at least 3
hours (longer is fine).
19. Discard the old TE buffer to the beaker and replace with 1L new sterile TE buffer. Stir
slowly for O/N at 4˚ C. The next morning, change TE buffer one more time and stir slowly for
another 3-4 hrs at 4˚ C.
Slowly pull BAC DNA out of the cassette by using 1ml or 3ml syringe with 21 gauge needle.
into a 15 ml conical tube. Add 2x volume of cold 100% ethanol (stored -20˚C freezer) and 1/10
volume of 3M NaOAC pH7.0 into each tube. Gently mix. Transfer the mixture into two equal
portions into two X 2ml eppendorf tubes by very gently pipetting. Keep the eppendorf tubes at
-20˚C for 30min. Spin down in a microcentrifuge at max speed at RT for 15 min. Decant
supernatant, invert the tube and tap briefly on paper towels. Add 100ul of cold (-20˚C) 70%
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Modified by Yue Hou March 2009
ethanol, spin down again for 2 min at RT. Remove all of 70% ethanol, air dry the pellet briefly
(about 3 min), then add 50 ul of filtered micro-injection buffer or TE (or more volume based on
the size of the pellet). Gently tap the bottom of the tube a few times. To dissolve the BAC DNA,
keep the tube at 4˚C at least O/N to allow BAC DNA to completely go into solution. (In most
time, my BAC DNA was kept at 4˚C in this step over the weekend before I used it to run CHEF
gel for BAC DNA qualification – Yue Hou, 2009). Gently tap the tube to help disperse the DNA
– do not pipette up and down to resuspend as you might for a plasmid DNA.
20. BAC DNA Quantification: the BAC DNA yield is usually 10-50 µg. We have found that
BAC preps give inaccurate UV spec readings. Instead, quantify by digesting with NotI or SalI to
excise the BAC vector band, and compare on EtBr-stained CHEF gel to lambda HindIII bands of
known quantity. (e.g. with BioRad QuantityOne software).
Note, the true total DNA concentration may be inflated by excessive sheared BAC or
contaminating bacterial DNA. That is, if the BAC DNA is estimated to be ~1 ng/µl based on
the gel bands, the total DNA conc. may be higher. This may cause embryo toxicity or needle
clogging during microinjection. In this case diluting the DNA to 0.5 ng/ul can help the injections
go smoother, though the rate of transgenesis may go down.
OPTIONAL: Some labs mix polyamines into the BAC DNA to stabilize it. Instructions for this
are attached as an appendix to this document.
OPTIONAL: 2009: We previously used Centriprep columns for some of the post-CsCl dialysis
steps . These allow concentration of the final DNA to some extent, but are labor intensive, so
this has been dropped from the current protocol. Instead the BAC DNA is precipitated and
resuspended for final cleanup. However a description of Centriprep column use for BAC
cleanup has been kept as an appendix. The dialysis and centriprep steps are somewhat
interchangeable. i.e. one can do more dialysis steps and omit the centriprep, or vice versa, with
similar results.
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Modified by Yue Hou March 2009
SOLUTIONS
Solution I (a.k.a. GTE) + RNASe A
final conc:
50 mM Glucose
25 mM Tris pH 8.0
10 mM EDTA
RNAse A 50 µg/ml
ddH20:
100 ml
500 ml
0.9 g
2.5 ml 1 M
2.0 ml 0.5 M
0.5 ml 10mg/ml
to 100 ml
4.5 g
12.5 ml 1 M
10.0 ml 0.5 M
2.5 ml 10mg/ml
to 500 ml
Filter-sterilize and store at 4˚ C.
Solution II
Make fresh each time.
final conc:
0.2 M NaOH
1 % SDS
100 ml
2 ml 10 M
10 ml 10%
88 ml ddH20
Solution III
1 liter
Dissolve 294.5 g Potassium Acetate in approx. 500 ml ddH20. Add glacial acetic acid to lower
pH to 5.5 (will require 150-200 mls). Bring final volume up to 1 liter with ddH20. Store at 4˚ C.
MIB (Microinjection buffer): 10 mM Tris, pH 7.4 / 0.15 mM EDTA pH 8.0, made up from
autoclaved 1 M Tris and 0.5 M EDTA stocks and tissue-culture-grade or embryo-grade distilled
water (e.g. Sigma cat #W1503-500ML), and filtered through 0.22 micron filter to remove any
fine particulates. Store at room temp.
TE/NaCl saturated butanol
Prepare and use in fume hood.
In 250 ml bottle (autoclave stir bar in bottle):
Add 35gm NaCl to 100ml TE.
Stir 15 min @RT to dissolve (most, though not all of NaCl will go into solution).
Add 100ml n-butanol, stir vigorously for 1 hr.
Store @ RT under fume hood (w/alcohols).
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Modified by Yue Hou March 2009
APPENDIX 1
Optional step: Polyamines buffer for BAC DNA microinjection
It has been shown that a buffer made with 100 µM polyamines (e.g. spermine and
spermidine) and 100 ml NaCL helps protect large DNA molecules from shearing. This can be
added to BAC DNA before microinjection, and can increase the frequency of transgenesis and
probably also the frequency transgene integrations having intact copies of the whole BAC
molecule.
1. Make 1000x Polyamines Stock and store aliquots at –20˚.
2. 1 week before the scheduled date for BAC pronuclear injection, dilute BAC DNA to 2 ng/µl
in microinjection buffer (MIB; see BAC DNA prep), using clean microfuge tubes that have also
been pre-rinsed with MIB. Make a 2x working dilution of polyamines buffer.
3. Combine equal volumes of 2 ng/µl BAC DNA and 2x polyamines buffer. Gently mix by
tapping the tube. Final solution will be 1 ng/µl BAC DNA and 1x polyamines buffer. This
incubation time enhances the complex formation between DNA and polyamines. Store at 4˚ C
for 1 week to allow DNA/polyamine complex formation. Then it is ready for microinjection.
1000x PA (Polyamines) Stock :
30 mM Spermine, molecular biology grade (Sigma, tetrahydrochloride, #S-1141)
70 mM Spermidine, molecular biology grade (Sigma, #S-0266)
Dissolve the spermine and spermidine together in sterile tissue-culture-grade distilled water,
filter sterilize (0.2 micron filters), and store the 1000x stock aliquots at -20 C. 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. Discard frozen stocks after 6 months.
2x Working dilution of PA (polyamines ) buffer:
Prepare this fresh and discard unused portion. You only need a small amount but it is convenient
to make a large amount.
To make 50 ml of 2x working dilution PAbuffer : prepare in sterile 50 ml tube pre-rinsed with
MIB or sterile water. Filter-sterilize before use.
Use this much stock solution
Final concentrations in the 2x PA buffer
1 ml of 1 M Tris-HCl, pH 7.5 (sterile)
20 mM Tris-HCl, pH 7.4
20 microliters of 0.5 M EDTA, pH 8.0 (sterile)
0.2 mM EDTA, pH 8.0
2 ml of 5 M NaCl (sterile)
200 mM NaCl
100 microliters of the1000x PA stock
200 nM polyamines
46.88 mls water*
-
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Modified by Yue Hou March 2009
* we use Tissue-culture-grade or “embryo grade” sterile distilled water (e.g. Sigma cat
#W1503-500ML).
References:
Polyamine buffer:
1. Montoliu, L. et al. 1995. J Mol Biol 246(4):486-492
2. Thom Saunders and U. of Michigan transgenic core web site,
http://www.med.umich.edu/tamc/index.html
3. Nagy et al. Manipulating the Mouse Embyro 3rd edition)
4. BAC transgenesis efficiency is helped by using the buffer:
(GENSAT project) Gong, S. et al, 2003. Nature, 425(6961):917-925.
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Modified by Yue Hou March 2009
APPENDIX 2
Optional step: Centriprep-YM30 buffer exchange
These columns can be used to perform “speed dialysis” and concentration of BAC DNA , for
example in place of Slidealyzer dialysis. However they are somewhat labor intensive.
To exchange the buffer for microinjection buffer (MIB) , centriprep-YM30 columns (Amicon
brand, sold by Millipore, cat.#4306) are used. Read the column instructions carefully before
using. Basic idea: The DNA is in the outer chamber, so after adding the DNA make sure to only
discard the inner chamber solution at each step. Make sure to not dump out the outer chamber
solution by accident.
All spins are at 2800 rpm at 4˚ C in a rotor designed to support 50 ml tubes. (Mortlock
lab: use F14S “Fiberlite” rotor.)
1. Prerinse column w/10 mls MIB, 10 min. Spin; discard both inner + outer chambers;
repeat.
2. Mix BAC DNA with 15 mls MIB, put in OUTER chamber, spin 20-25 min.
3. Discard INNER chamber, spin 15-20 min. Repeat spin twice more & discard INNER
chamber each time.
4. Add 10 mls MIB to OUTER chamber, spin 10-15 min,
5. Discard INNER chamber liquid, spin again for 10-15 min, & discard INNER soln.
again.
6. Repeat steps 4-5, 3x. (note: do this 7x If you did not do the dialysis in step 18 of the
main protocol.)
7. Purified BAC DNA in MIB solution remains in the OUTER chamber (should be <0.5
ml volume; if not, repeat spins until it is.). Carefully transfer the BAC DNA into a
microcentrifuge tube for storage at 4˚. Do not freeze/thaw BAC DNA.
By the centriprep column method, the concentration of BAC DNA you can get is about 1050ng/ul from 0.5-1 L of culture.
8