U DNA mutagenesis

skspoidn at reading.ac.uk skspoidn at reading.ac.uk
Sat Jun 5 07:04:53 EST 1993


Well, 6 requests for my protocol later probably justifies a posting here, so here we go.

GROWTH OF PHAGE
1. Inoculate 20ml LB broth in a 50ml flask with 10ul of O/N culture of CJ236
	Incubate with shaking at 37

2. 6 hours later add 1-3 x10E6 phage and incubate O/N at 37 with shaking

3. Remove ALL the cells with your favourite centrifuge at 10000rpm
	 for 10min. Do this twice if need be.

4. PEG precipitate (.25 vol 3.5M ammonium acetate, 20% PEG8000 on ice for 30min)
	Use a polyallomer tube as PEG does not stick to polycarbonate

5. Pellet PEG ppt, drain off all supernatant, resuspend in 200ul TE

6. Titre phage on MV1190 (or whatever f+ strain you like) and CJ236 cells.
 If there is not at least a 4 log reduction in titre on the MV cells, start again.


EXTRACT THE DNA
use your fave extraction procedure here. I use 
repeated phenol/CHCl3 extraction and EtOH ppt

ANNEALING OF PRIMER
put in a tube:
U DNA template	100ng
oligo		100 fmol
10xanneal buf.  1ul
water		to 10 ul

incubate at 65 deg for 5 min, followed by room temp for 10-15 min.

10x annealing buffer=200mM Tris pH 7.4, 20mM MgCl2, 500mM NaCl

Note that there should be a ratio of primer:template of between 2:1 and 10:1.


SYNTHESIS OF COMPLEMENTARY STRAND

Put in a tube:
Annealing soln from above	10ul
Synthesis buffer		1ul
T4 DNA ligase			2ul (2-4 units)
SS binding protein		1ul (1ug)
T4 DNA Pol			.5ul (1-2.5 units)

1. Incubate on ice for 5 min followed by 37 for 30 min

2. Add 40 ul TE buffer and terminate reaction by freezing.
	 solution is stable for at least 1 month.

3. Run 10ul of reaction on 1% agarose gell to monitor DNA synthesis.

4. Transform 1-3ul of solution into MV1190's, pick plaques next day etc etc.

10x synthesis buffer is 5mM dNTPs, 10mM ATP, 100mM Tris pH 7.4, 50mM MgCl2, 20mM DTT

phew.

Mike Poidinger
Dept of Microbiology
University of Reading
Ok, so it's not a kit, but the exception proves the rule.



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