Single-Stranded Rescue Procedure

[Karl Fischer]

In article <350A0E4D.CC253AED at usa.net>, netson at usa.net wrote:

>         I would like to convert a cDNA clone in pBluescript vector to
> single-stranded DNA by VCSM13 helper phage.  Do you have the
> single-stranded rescue procedure for VCSM13 helper phage?  I will be
> very grateful if you can post or email the procedure to me.

Posted and emailed.
Wilson,

Please find below my protocol for generation of ssDNA from a phagemid; it
uses M13K07 instead of VCSM13 so adjust antibiotic selection appropriately
(can't recall at this time of night if VCSM13 is kan R). 

All the best.

Karl the hepB guy

Growth of E. coli to maintain the F pilus

In order to maintain the expression of the F pilus so as to infect with a
M13 virus (wt or variant), the E. coli is grown in a modified M9 medium.

Stock solutions

10X M9 salts      NaHPO4 € 7H20     60 g
                  KH2PO4            30 g
                   NaCl              5 g
                  NH4Cl             10 g

Dissolve in 900 ml dd water and adjust pH to 7.4 with NaOH. Autoclave.

1 M MgSO4            autoclave
100 mM CaCl2         autoclave
30% glycerol (in water)    Filter through 0.45 µm.
10% Thiamine         Filter through 0.45 µm.
10% Casamino acids (in water) autoclave

To prepare a 5 ml broth :

0.5 ml   10X M9 salts
0.1 ml      30% glycerol
0.1 ml      10% casamino acids
5 µl     1 M MgSO4
5 µl     100 mM CaCl2
1 µl     10% Thiamine
4.3 ml      ddH2O


Modified Methodology for Infecting Phagemid-Containing E. coli

Grow cells O/N in M9 medium (5 ml culture is sufficient for most purposes)
under antibiotic selection for maintenance of the phagemid.

The next day transfer a 1ml aliquot of the culture to a 50 ml Falcon tube. 
Inoculate with the helper phage (M13 K07 or R408) at an moi of 10 (approx.
1 X 1010 pfu). 
Place at 37°C with gentle rocking for 1 hour. 
Add 19 ml of medium (2X YT, LB) with kanamycin sulfate added to 
70 µg/ml if M13K07 is used. 
Place tube(s) in a 1 litre flask and shake vigorously O/N at 37°C.

Do not include kanamycin if R408 is used !!!!!!!!

Pellet cells at 3,000 rpm, 15 minutes. 
Decant supernatant to fresh 30 ml Corex tube.
Centrifuge at 10,000 rpm, 5 minutes. Carefully remove supernatant to a
fresh Corex tube.
Precipitate phage by adding 0.25 volumes 20% PEG 8000-2.5 M NaCl. Mix and
let stand on ice for „1 hour. 
Pellet at 10,000 rpm, 15 minutes, 4 °C.
 Aspirate supernatant. Remove remaining fluid with cotton swab. 
Resuspend pellet with 0.4 ml of TE or ddH2O. Transfer to an Eppindorf tube
and extract with an equal volume of chloroform (vortex 1 minute,
centrifuge 2 minutes). 
Extract aqueous phase with phenol:chloroform (1:1) until no precipitate is
visible at interface. 
Add 0.5 volume of 7.5 M ammonium acetate and 2 volumes of ethanol. 
Chill at ­20°C for „ 1 hour. 
Pellet DNA by centrifuging for 15 minutes at 4°C.
Aspirate the supernatant, wash the pellet with 70% ethanol.
Air-dry the pellet and resuspend in 20 µl ddH2O.
Run a 4 µl aliquot on a gel with suitable size markers to evaluate purity
and quantity of DNA.

Sizes of some ssDNAs

M13 mp19    7.25 kb
M13 K07     8.7   kb
R408        6.4   kb