Identifying Trangenic Founders

 

IDENTIFYING TRANSGENIC FOUNDERS (by Dr. Katya Ravid with members of the core)

OVERVIEW

When your litters of potential founders are born you now must determine how you will screen for founder animals. The two most commonly used methods for transgene detection are Southern Blot and PCR analysis. The DNA used for the Southern Blots usually is derived from the animal’s tail, the DNA used for PCR is usually from tail or blood. An alternative to both of these methods is Dot Blot analysis, but this method is not recommended for screening founders due to the frequency of both false positives and false negatives. Tail DNA can be particularly difficult to isolate and digest. When isolating DNA from tails you should use a very rigorous procedure, to assure the digestion of the tail and the purity of the DNA. When using PCR to screen your animals it has been shown that the cleaner the DNA the less likely you are to experience false positives and negatives. Southern Blot analysis has been shown to be the most reliable method for screening founder animals. The following protocols for DNA isolation from tails have been used successfully for Southern Blots and have had the most success with PCR analysis.

ISOLATING GENOMIC TAIL DNA

PHENOL / CHLOROFORM METHOD

Cut 1-2 cm of tail and place it into a 1.5 ml micro centrifuge tube, mincing the tail is not necessary.

Add 500 µl of tail digestion buffer containing 0.5 mg/ml of proteinase K.

Incubate overnight at 55 o C, (in a water bath, oven, heat block, or anything that will hold the temperature constant).

Remove the tubes from 55 o C. Add 500 µl of phenol and vortex vigorously to mix the phases completely. Centrifuge for ten minutes at speed rpm / rcf g x 10, 1250 and transfer the top 600 µl of the aqueous phase, or to just before the interface, to a fresh 1.5 ml micro centrifuge tube. Repeat this step if aqueous phase is cloudy.

Add 500 µl of phenol / chloroform and vortex vigorously to mix the phases completely. Centrifuge as above for ten minutes and again transfer the top 600 µl of the aqueous phase, or to just before the interface, to a fresh 1.5 ml micro centrifuge tube.

Add 500 µl cold of isopropyl alcohol and vortex vigorously. DNA should immediately form a stringy precipitate.

Centrifuge as above for ten minutes to pellet the DNA, then remove and discard as much of the supernatant as possible.

Rinse the pellet with .25 m l of 80% ethanol (room temperature). This step is to remove any traces of salts or phenol.

Air dry the pellet or use a speed vac, and re -suspend the DNA in 60 µl of TE, (T 10E 1). Use 10 – 20 µl of each DNA for your Southern Blot analysis.

* You can reduce the digestion time to 4-6 hrs. by increasing the proteinase K concentration (no greater than 0.75 mg/ml) and by mixing the tails periodically during the digestion.

? Tail Digestion Buffer: 100 mM NaCl TE: 10mM Tris, pH 7.6
10 mM Tris, pH 7.6 1mM EDTA, pH 8.0
25 mM EDTA, pH 8.0  
0.5% SDS  

ALTERNATIVE PROTOCOLS FOR PREPARATION OF GENOMIC DNA

The following is an alternative protocol that is somewhat similar, and also yields a similar amount of DNA that can be digested with most restriction enzymes [9]. Although this method works fine with Southern Blots it is not as reliable when used with PCR.

Cut 1-2 cm of tail and place it into a 1.5 ml micro centrifuge tube, mincing the tail is not necessary.

Add 700 µl of tail digestion buffer to each tube along with 35 µl of a 10 mg/ml solution of proteinase K.

Incubate overnight at 55 o C, in a water bath or in an oven on a rocking platform.

Remove the tubes from 55 o C. Add 700 µl of phenol and shake vigorously to mix the phases completely.

Centrifuge for three minutes, so that phases separate.

Transfer the aqueous phase to a fresh 1.5 ml micro centrifuge tube, being careful not to pick up any phenol or material at the interface.

Add 700 µl of phenol / chloroform (1:1), and shake vigorously for two minutes, and centrifuge for two minutes.

Again transfer the aqueous phase, avoiding the interface, to a fresh 1.8 ml micro centrifuge tube.

Add 70 µl of 3M sodium acetate (pH 6), and 700 µl of 100% ethanol at room temperature. Shake to mix thoroughly. DNA should immediately form a stringy precipitate. A sodium acetate solution with a pH lower than 6 will cause the EDTA to precipitate.

Spin in a micro centrifuge for 30 seconds to pellet the DNA. Remove and discard as much of the ethanol supernatant as possible.

Add 1ml of 70% ethanol (room temperature), to each tube, vortex or shake vigorously to wash the pellet. This step is essential to remove any traces of SDS and phenol.

Spin in a micro centrifuge for one minute at room temperature. Remove as much of the ethanol supernatant as possible. Air-dry or use a speed vac.

Add 100µl of TE to each tube. Leave at room temperature overnight. To dissolve the DNA pellet quickly you can put it at 65 o C for 10 -15 minutes. Use 10 -20 µl of each DNA for your Southern Blot analysis.

? Tail Digestion Buffer: 50 mM Tris, pH 8.0 TE: 10mM Tris, pH 8.0
100 mM EDTA, pH 8.0 1mM EDTA, pH 8.0
0.5% SDS  

SODIUM CHLORIDE METHOD

An alternative for anyone who does not wish to work with phenol or chloroform or would just like a quicker protocol for isolating tail DNA is the NaCl method. The DNA obtained from this protocol is clean enough for Southern Blots or PCR.

Cut 1-2 cm of tail and place it into a 1.5 ml micro centrifuge tube; mincing the tail is not necessary.

Add 750 µl of tail digestion buffer to each tube containing 0.5 mg/ml of proteinase K and incubate overnight at 50 o C.

Remove the tubes from 50 o C. Add 400 µl of 6M (saturated) NaCl and shake vigorously ( 200 times) in a rack (do not vortex). Place on ice for 10 minutes. Spin in a micro centrifuge for 10 minutes.

Squirt 1 ml of supernatant into 2 ml of ethanol in a 4 ml tube. Do not mix. In 5-10 minutes, the DNA will form a stringy precipitate that floats above the layer of salt and can be lifted out with a pipet tip and transferred into a fresh micro centrifuge tube.

Air dry 5-10 minutes.

Re-suspend the DNA in 400 µl of TE (T 10E 1). Let the DNA stand at room temperature for 24 hours before using . To dissolve the DNA pellet more quickly , you can put it at 55 o C for 1-2 hours or at 37 o C for 4-6 hours.

* DNA isolated in this manner can at times be difficult to go into solution, therefore reducing the quantity of DNA recovered.

? Tail Digestion Buffer: 20 mM Tris, pH 7.6 TE: 10mM Tris, pH 7.6
100 mM EDTA, pH 8.0 1mM EDTA, pH 8.0
0.5% SDS  
100mM NaCl  

Tail DNA prepared using one of these protocols or any variation will contain a substantial amount of RNA, but this will not interfere with the restriction enzyme digest or the Southern Blot analysis. When preparing for restriction enzyme digestion, add 5 µg of DNase-free RNase A to each sample along with the restriction enzyme. Digestion with certain restriction enzymes may also be aided by adding ~ 5 µl of 4mM spermidine to the reaction [9]. Digest one-tenth of the DNA per lane on Southern.

Boston University School of Medicine/Transgenic Knock Out Core Facility


Scientific Director of Transgenic/Knock out Core, Dr. Katya Ravid
Transgenic Specialist and Manager, Greg Martin

Primary teaching affiliate
of BU School of Medicine