Following Mass Excission the transfection efficiency of the
phagemid Bluescript stock, packaged as filamentous phage particles,
needs to be determined by titration against freshly grown SOLR cells.
This is due to the high efficiency of the excision process and the
requirement to achieve single-colony isolation for archiving and
screening.(This stock should be stable for up to 6 months at
4¡C.)
Preparation Of Host Cells
DAY 1-Prepare SOLR colony streaked plates
Revive the host cells by streaking from ice splinters from
frozen bacterial glycerol stock with a sterile loop onto plates
containing LB agar and appropriate antibiotic.
_____________________________________________________________________________________
Bacterial Agar plate for
Medium for Medium for
bacterial cultures for
strain bacterial
streak glycerol stock titering phage (final concentration)
_____________________________________________________________________________________
SOLR strain
LB-kanamycin LB-kanamycin LB
without supplements
_____________________________________________________________________________________
DAY 2-Prepare overnight SOLR cell cultures
Bacterial cultures should be started from a fresh colony plate
(<7days old) using a single colony.
1 Inoculate 50ml of LB broth (without supplements) with a single
colony of SOLR cells.
2 Grow overnight cultures of the SOLR cells with vigorous shaking at
30 -37 C.
The lower temperature ensures that the cells will not overgrow.
3 Prepare 16 LB-ampicillin4 agar plates
DAY 3-Titration of phagemid stock: using freshly
prepared SOLR cells.
1 Gently spin down the SOLR cells (1000 x g for 10
minutes).
2 Resuspend the cells in ~15 ml of 10 mM MgSO4. Do not vortex.
3 Dilute SOLR cells to an OD600 of 1.0 in 10 mM MgSO4.
Highest efficiencies are obtained from freshly prepared cells.
4 Prewarm and dry LB-ampicillin agar plates ready for plating in step
9
5 Make x2 working dilutions of phagemid stock => 1:10 and 1:1000
with LB broth
6 Add 400 microl of freshly grown SOLR cells from step 3 (OD600= 1.0)
to x8 1.5ml tubes.
7 Add working dilutions of phagemid stock to each tube as follows
(volumes to add in microlitres)
|
phagemid stock dilution |
tube # |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
|
1:10 |
|
20 |
10 |
2 |
1 |
|
|
|
|
|
1:1000 |
|
|
|
|
|
20 |
10 |
2 |
1 |
8 Incubate the tubes at 37 C for 15 minutes.
9 Add 200 microl from each tube to replicate LB-ampicillin agar
plates (50 microglml)
and distribute evenly over plate surface with sterile glass
spreader
Allow excess media to dry before inverting plates
10 Incubate overnight at 37 C.
Colonies appearing on the plate contain the Bluescript
double-stranded phagemid with the cloned DNA insert. Helper phage
will not grow, since helper phage is unable to replicate in Su-
(nonsuppressing) SOLR strains and does not contain
ampicillin-resistance genes. SOLR cells are also resistant to lambda
phage infection, thus preventing lambda phage contamination after
excision.
DAY 4-DETERMINATION OF TITRE OF MASS EXCISSION
SUPERNATANT
Calculation of titre
|
tube # |
microlitres of phage |
number of |
factor |
titer per ml |
total number |
|
1 |
1 |
|
1 x 10 e3 |
|
|
|
2 |
0.5 |
|
2 x 10 e3 |
|
|
|
3 |
0.1 |
|
1 x 10 e4 |
|
|
|
4 |
0.05 |
|
2 x 10 e4 |
|
|
|
5 |
0.01 |
|
1 x 10 e5 |
|
|
|
6 |
0.005 |
|
2 x 10 e5 |
|
|
|
7 |
0.001 |
|
1 x 10 e6 |
|
|
|
8 |
0.0005 |
|
2 x 10 e6 |
|
|
LB BROTH (PER LITER)
10g of NaCl
10g of bacto-tryptone
5g of bacto-yeast extract
Adjust to pH 7.0 with 5 N NaOH
Add deionized H2O to a final vol. of 1 liter
Autoclave
Allow medium to cool to 55 C before addition of antibiotic
LB AGAR (PER LITER)
10g of NaCl
10g of bacto-tryptone
5g of bacto-yeast extract
15g of bacto-agar
Adjust pH to 7.0 with 5 N NaOH
Add deionized H2O to a final vol. of 1 liter
Autoclave
Allow medium to cool to 55 C before addition of antibiotic
Pour into petri dishes(~25 ml/100-mm plate)
[Kanamycin] 50 microg/ml
[Ampicillin] 50 microg/ml