Method 2

The protocol below has proven effective at providing high quality DNA for pronuclear microinjection. In addition, the TMS also recommends the use of QIAGEN QiaQuick and QiaEX II kits for cleanup of electrophoretically separated transgenic cassettes.

This method is kindly provided by one of our users

Phenol Extraction of DNA Fragment from Low Melting Point Agarose (all at room temp)

1. Cut 25ug of DNA using appropriate restriction enzymes and separate insert and vector by electrophoresis in low melting point agarose (FMC brand)
2. Cut out band using razor blade taking care to minimize gel volume and weigh. Add equal volume of 0.3M NaCl/TE and heat at 65-70°C for 30 min.
3. Extract with one volume of Tris-buffered Phenol. Spin for 10 min in microfuge and take aqueous phase. Back extract with 1/2 volume of 0.3M NaCl/TE.
4. Pool aqueous phases and extract with phenol/chloroform (Repeat this step if aqueous phase is not clear after this step).

Purification on PrePac Column (BRL)

(Allow all solutions to flow through column by gravity flow)

Hydrate column	2ml of 1M NaCl/TE
Equilibrate column	2ml of 0.2M NaCl/TE
Load Sample	DNA in 1ml 0.2M NaCl/TE
Wash sample	3ml of 0.2M NaCl/TE
Elute sample	3 lots of 100 µl 1M NaCl/TE

Precipitate DNA by adding 2 volumes of EtOH. Wash pellet once with ice-cold 70% EtOH. Dry pellet in speed-vac. Resuspend in 100 µl of DNA Injection Buffer.

N.B. For large fragments the following is recommended:

Hydrate column	3ml of 2M NaCl/TE
Equilibrate column	3ml of 0.2M NaCl/TE
Load Sample	DNA in 1ml 0.2M NaCl/TE
Wash sample	3ml of 0.2M NaCl/TE
Elute sample	4 lots of 100 µl 1M NaCl/TE

Dot Dialysis

1. Add several ml of DNA Injection Buffer to a disposable petri dish.
2. Float a 0.1um Millipore filter on buffer and leave for 5 min to equilibrate.
3. Gently pipette DNA solution onto filter and leave for 1.5-2 hrs. Remove DNA solution and attempt to recover as much volume as possible. Add half the volume of solution recovered to filter, leave for several min and recover this sample also (improves yield).
4. Spin DNA solution for 30 min in microfuge and take ONLY TOP 90% of solution (this step is very important to remove particulates that will block injection pipette, failure to adequately remove particulates may result in having your DNA sample returned to you for re-purification).
5. Dilute an aliquot of DNA solution to close to the final concentration of approx. 5 ng/µl and estimate concentration exactly by comparison with known standards on an agarose gel.

Solutions for Method 2

(Sterilize by filtration)

DNA Injection Buffer:

Stock	For	100 ml	Final Conc
Tris (pH 7.4)	1M	1ml	10mM
NaCl	4M	250µl	10mM
EDTA	1M	25µl	0.25mM

1M NaCl/TE:

Stock	For	10ml
NaCl	4M	2.5ml
TE (pH7.6)		7.5ml

0.2M NaCl/TE:

Stock	For	10ml
NaCl	4M	500µl
TE (pH7.6)		9.5ml