Single-stranded DNA prep reference?

Anders B. Jensen abjensen at biobase.dk
Tue Jun 13 04:41:18 EST 1995


Preparation of single stranded DNA template

This protocol has been used for oligonucleotide-directed mutagenesis of
a template in which a small number of uracill residues occupy the
appropriate positions in place of thymidine. The uracil-containing DNA
is produced in an E. coli dut- ung- (CJ236) strain lacking the enzymes
dUTPase and N-glycosylase. Consequently, in this strain, deoxyuridine is
incorporated into DNA in place of thymidine and is not removed. However,
the procedure can be used for all M13 based phages.

1)  Transform CJ236 (dut- ung-) with the phagemid containing the insert
of interest and the M13 origin (like pBluescript).
2)  Grow a overnight culture of the transformed strain in LB/amp+cam (to
maintain the plasmid and F' episome).
3)  Add 1-2 µl VCSM13 (kanr) helper phage and incubate at 37 oC for 15
min.
4)  Add this to 200 ml LB/amp and grow for 1 hour.
5)  Add kanamycin to 25 µg/ml and continue to grow for 16-24 hours, or
until growth has reached saturation (do not add cam in this step, since
cam inhibits sDNA replication).
6) Centrifuge for 15 min at 6000 rpm to pellet bacteria.
7)  Transfer the supernatant to a fresh centrifuge bottle, and add 40 ml
20% PEG(MW 6000)/2,5 M NaCl. Mix well and put on ice for 2 h.
8)  Centrifuge for 20 min at 6000 rpm to pellet phage particles.
9)  Decant the supernatant carefully, but leave behind a few ml. Use
this few ml to resuspend the phage pellet and transfer it to a corex 30
tube.
10)  Centrifuge the corex tube for 10 min at 8000 rpm to pellet phage
particles again, and carefully remove all the supernatant by aspiration.
11)  Dissolve the pellet in 1 ml TE + 20 µg/ml Rnase and transfer the
solution into two microfuge tubes and incubate fo 30 min at 37 C.
12)  Centrifuge for 5 min to remove any remaining cells. Transfer the
supernatant to fresh tubes.
 13)  Add 100 µl 20% PEG/2,5 M NaCl, mix well and leave to stand for 15
min at room temperature.
14)  Centrifuge for 5 min and carefully remove all remaining traces of
PEG.
15)  Resuspend the viral pellet in 500 µl TE buffer and add 500 µl
phenol saturated with TE buffer. Vortex 15-20 sec.
Centrifuge for 5 min and transfer the upper phase to a new tube. Repeat
this step with phenol/chloroform/isoamyl alcohol until the interface is
clean (2-5 times).
16)  Repeat twice only with chloroform.
17)  Precipitate the DNA with 1/10 volume 3 M NaAc and 1 volume
isopropanol.
18)  Wash the pellet with 70% EtOH and resuspend each tube in 100 µl TE.






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