Identification of the bcd1-2 allele

Tetrahymena Fast DNA Extraction

Prepared August, 1993

References:     

Martin Zillman, personal communication

Gaertig J, Thatcher TH, Gu L, Gorovsky MA. Electroporation-mediated replacement of a positively and negatively selectable beta-tubulin gene in Tetrahymena thermophila. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4549-53. doi: 10.1073/pnas.91.10.4549. PMID: 7910408; PMCID: PMC43823.

This procedure makes total DNA that can be used for PCR or Southern blotting. Volumes for a 1.5 ml miniprep are given in bold.

1). Grow cells to mid log phase (2-4 x 10⁵ cells per ml). Starve 15-25 mls of cells in 10 mM (60 mM if using F2 pools for extraction of DNA for NGS) Tris-HCl pH 7.5 for 18-48 hrs. 

For NGS preps, I starve for 2 days at the room temperature.

2). Spin down 15-25 mls of cells at 3000 rpm 5 minutes (1.5 ml in a microfuge tube 30 sec. 3-6K rpms, not too fast, you do not want to break cells)

3). Wash with 15 ml 10 mM Tris-HCl pH 7.4, spin again.

2) Remove the supernatant leaving not more than 0.3 ml (50 µl). Resuspend the pellet by taking it into and out of the (glass Pauster pipette or transfer pipet). Add up to 5 ml of urea buffer (0.7 ml, leave in microfuge tube). Gently shake until homogeneous.

3)   Extract the lysate twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform:isoamyl alcohol (24:1) with gentle shaking (no vortexing). Spin the sample between extractions at 3500 rpm 10 min at 10^oC. During each extraction recover the top aqueous layer.

Regarding the temperature, I usually spin in cold temperature (e.g. 10-15^oC) but sometimes at the room temperature and I did not notice a difference (JG).

4)   Add 1 ml (0.15 ml) 5 M NaCl to the approximately 3 ml (0.5 ml) of lysate recovered. If you recovered a different volume, adjust the volume of 5 M NaCl accordingly. (The addition of salt helps to reduce the carbohydrate content of the final precipitate.)

5)   Precipitate DNA with an equal volume of isopropyl alcohol. If there is enough of DNA (a lot of strands visible), spool the DNA onto a glass pipet, transfer to an Eppendorf tube, wash with 70% ethanol and dry in a speed vac. Alternatively, let DNA settle to the bottom of the 15 ml tube tube and remove the supernatant using a Pauster pipet, wash with 70% ETOH, settle the DNA, remove the sup and dry in a speed vac. If there is only a small amount of DNA present in a 15 ml tube, spin down as above in cold to get a pellet.  On a miniprep scale spin down in a microcentrifuge in cold (10-12K rpms) for a few minutes. 

6)  Wash with 70% ethanol if you have not done it earlier. Remove as much of the alcohol supernatant as possible using a yellow pipet tip. Dry the pellet (or DNA clamp) in a speed vac. On the large scale, if you were not able to spool and DNA is still in a 15 ml conical tube, remove the speed vac rotor, place the sample inside the speed vac chamber (use the plastic tube rack left near the speed vac to keep the tubes in position while under vacuum).

7)   Resuspend DNA in 400-600 µl (20 µl) TE and add 4-6 µl (1 µl) RNAse A (10 mg/ml). Incubate at 55°C overnight in the water bath (for minipreps 1 hr at 37^oC is enough). After 1 hr use a blue tip (cut at the end to avoid shearing) transfer the DNA sample to an Eppendorf tube and continue to incubate at 55^oC overnight. 

8)   Pelletable carbohydrates can be removed by centrifugation at 31,000 x g for 45 minutes at 20°C (35,000 rpm in the tabletop ultracentrifuge). I have not been doing this for a long time and the resulting DNA is good enough for PCR and NGS library construction.

The yield is typically 100-300 mg/ml. 

Preparation of the urea buffer.

1. Prepare a “2X no urea” buffer as follows. The buffer can be stored indefinitely at the room temperature but SDS will precipitate:

   2X buffer no urea (100 ml):

   14 ml 5 M NaCl

   2 ml 1 M Tris pH 7.4

   4 ml 0.5 M EDTA

   20 ml 10% SDS

   H2O to 100 ml

2. On the day when you need to prepare the 1X urea buffer, warm up the bottle with the 2X no urea buffer (e.g. heating for a few sec in a microwave oven but make sure that the cap is completely loose or the bottle could explode). 
3. Weight 4.2 g of urea.
4. Measure out 5 ml of the warmed up 2X no urea buffer into a 15 ml tube, add 4.2 g of urea, add water to 8-9 ml, warm up the solution with running tap water, and when nearly dissolved adjust the final volume to 10 ml. This is the 1X urea buffer.