TELT Microquick

This method, if carried out carefully, should result in DNA which can be sequenced with the Applied Biosystems cycle sequencing kit. The yield should be about 50 µg plasmid DNA per preparation (if you handle well growing pUC derivatives). You only need one (a single) tube per sample! And the whole method per sample costs only 3% of a usual column-kit!

- grow bacteria (E.coli strain XL1-blue) over night with good aeration (note 1) in 3 ml TB-medium (see below),
- fill 1.5 ml of the culture in a labelled Eppendorf tube,
- centrifuge (2 min, full speed) (note 2),
- remove the medium completely - the pellet should be dry (note 3),
- resuspend cell pellet in 200 µl TELT + 20 µl Lysozyme solution completely,
- incubate for 3 min at 95 °C (note 4),
- incubate for 5 min on wet ice,
- centrifuge (15 min, 4 °C, 13.000 rpm),
- remove the slimy pellet with a clean toothpick entirely,
- leave the supernatant in the tube, add 100 µl isopropanol and mix,
- centrifuge (15 min, 4 C, 13.000 rpm),
- discard supernatant, wash pellet with 200 µl 70% ethanol,
- centrifuge briefly and remove residual liquid completely,
- solve pellet in 50 to 100 µl TE/R (note 5),
- use 2 µl for one restriction digest.

Once candidates for sequencing have been identified, proceed as follows:

- to the DNA solution add water to 100 µl final volume,
- add 100 µl 4 M NH4Ac (ammonium acetate) and mix,
- add 600 µl ethanol at room temperature,
- centrifuge (15 min, 4 C, 13.000 rpm),
- wash pellet thoroughly with 200 µl 70 % ethanol,
- centrifuge briefly and remove residual liquid completely,
- solve DNA in 10 mM Tris/HCl pH 8.0,
- quantify carefully (OD260 = 1 is equal to 50 µg/ml).

TB-medium

TB I: 0.17 M KH2PO4 (23 g)
0.72 M K2HPO4 (164 g), ad 1000 ml with water, autoclave.
TB II: 12 g Tryptone (or Peptone)
24 g Yeast Extract
4 ml Glycerol, ad 900 ml with water, autoclave
- prior to use mix 1 part TB I with 9 parts TB II.

TELT

50 mM Tris/HCl pH 7.5
62.5 mM EDTA (stock solution: pH 8)
2.5 M LiCl
0.4 % Triton X-100

Lysozyme solution

10 mg/ml Lysozyme
in 10 mM Tris/Cl pH 7.5 and 0.1 mM EDTA (TE 10/0.1)

TE/R

10 µg/ml RNase (from an stock which had been heated to 100 °C for 10 min)
in 10 mM Tris/Cl pH 7.5 and 0.1 mM EDTA (TE 10/0.1)

Notes

Note 1
Prior to the collection of the cells no lysis of the culture should have happened! Such lysis is observed if a culture grown in rich medium (like TB) in the stationary phase is allowed to sit without aeration. Already a few lysed cells cause the contamination of your plasmid DNA with genomic DNA - and that results in a lot of other problems.
Note 2
If you prefer to use L-Medium or similar media, you can collect cells from 3 ml of your culture to increase yield.
Note 3
We recommend to use an Eppendorf cristal tip connected to vacuum.
Note 4
A heating block is recommended - make sure that tubes are properly closed.
Note 5
The pellet may be located as a smeer on one side of the tube. Solve the DNA on a shaker.

last modified 27.02.98 by Bernd Weisshaar