Teeny prep Genomic DNA Isolation Method
Last updated:December 2, 2003 by Siew-Loon Ooi

 

Transformant harvest for genomic DNA Isolation

1) To harvest the transformants from 10 150 mm plates, prepare about 40 ml of water or 15% glycerol. Use 15% glycerol if the transformants are to be frozen.
2) Pour ~4 ml of water or 15% glycerol into each big plate.
3) Use the glass spreader to scrape cells to one corner of the plate. Tilt the plate by resting the plate on a 25 ml pipet or an eppendorf tube rack.
4) Use 5 ml pipet to transfer cell suspension into a 50 ml Falcon polypropylene tube.
5) Repeat steps 2-4 with all the plates, and transfer all the transformants into the same 50 ml tube.
6) Mix well by vortexing. Aliquot ~ 1 ml into an Eppendorf tube for genomic DNA isolation.
7) Freeze the remainder in 15% glycerol at –70¾C, if desired.

Genomic DNA Isolation

1) Thaw the transformants (if frozen). Aliquot a total of 12 OD600 into an eppendorf tube. For each sample, use a total of 12 OD600 to prepare genomic DNA.
2) Add 1 ml of 1 M Sorbitol/0.1 M EDTA to the cells.
3) Spin at top speed for 10” to collect pellet.

Cell lysis

4) Resuspend pellet in 300 µl of 1 M Sorbitol/0.1 M EDTA + 14 mM b-ME + 0.4 mg/ml zymolyase. The mixture is prepared as follows:

Stock Reagent Volume (µl) Final [ ]
1 M Sorbitol/0.1 M EDTA 300 ~1M Sorbitol/0.1M EDTA
14.3 M b-ME 0.35 14 mM b-ME
2.5 mg/ml zymolyase (20 T) 65 0.44 mg/ml of 20T zymolyase
Total Volume 365.35  


5) Incubate at 37¾C for 20’ to 30’ for zymolyase treatment. One can check using a microscope to ensure that spheroplasting is complete. Spheroplasted yeast cells look much darker than unspheroplasted yeast cells.
6) Spin at top speed 30".
7) Resuspend pellet in 400 µl of TE. If spheroplasting worked, the pellet is very sticky and difficult to resuspend.
8) Add 100 µl of lysis buffer to each tube. Mix by inverting the tube ten times. The lysis buffer is prepared as follows:

 

Lysis Buffer Volume for 26 Rxn (ml) Volume per Rxn (µl) Final [ ] including 400 µl TE
0.5 M EDTA, pH 8.0 1.5 ml 55.5 55 mM
2 M Tris, pH 8.0 0.6 ml 22.2 89 mM
10% SDS 0.6 ml 22.2 0.44%
Total Volume 2.7 ml 100  


9) Incubate at 65¾C for 30’.
10) Add 100 µl of 5 M KoAC, mix well.
11) Incubate at 4¾C for 1 hour or overnight.
12) Before centrifuging, add 50 µl of chloroform to each reaction. Mix well. The addition of chloroform helps to separate the supernatant from the white glue at the bottom of the Eppendorf tube. Just do it.
13) Spin at top speed at 4¾C for 15'.
14) Transfer supernatant (~ 600 µl) to a new tube.
15) Add equal volume (~600 µl) of 100% EtOH.
16) Spin at top speed at 4¾C for 10'.
17) Wash in 50% EtOH.
18) Incubate at RT for 5'.
19) Resuspend in 400 µl of TE buffer.

RNase A treatment and ethanol precipitation of genomic DNA

20) Add 12.5 µl of 2 mg/ml RNase A.
21) Incubate at 37¾C for 30'.
22) Extract with equal volume (400 µl) of phenol:chloroform.
23) Extract with equal volume (400 µl) of chloroform.
24) Precipitate with 120 µl of 7.5 M NH4OAc and 1 ml of 100% EtOH.
25) Set at –20¾C for 20’.
26) Spin at top speed at 4¾C for 15'.
27) Wash in 70% EtOH.
28) Air dry.
29) Resuspend in 50 µl of TE buffer.
30) Determine the concentration of the genomic DNA.
31) Resolve 2-3 µl of genomic DNA on a 1% agarose gel to determine the quality of the preparation. The entire genomic DNA preparation should run as one discrete band larger than 20 kb in size.

Comments


In comparison to the glass bead method, teeny prep genomic DNA isolation method yields genomic DNA of higher quality. However, this method is also more time consuming. From our experience, genomic DNA isolated from both methods worked, and it is important to not use too much cell pellet for each genomic DNA preparation reaction. A total of 10 to 30 OD600 cell pellet should be fine.