Long Flanking Homology (LFH-)PCR
Step 1 : Choosing a cassette and designing primers:
up fwd – about 1 kb upstream of the gene / region to be knocked out (about 20-25 nt)
up rev – within 10-50 bp downstream of the start of the gene to be knocked out.
At the 5’ end of this oligo add the sequence corresponding to one end of the cassette being used from the primer table.
do- fwd - within 10-50 bp upstream of the end of the gene to be knocked out.
At the 5’ end of this oligo add the sequence corresponding to the other end of the cassette being used from the primer table.
do rev – about 1 kb downstream of the gene to be knocked out (about 20-25 nt).
* If an upstream or downstream gene overlaps the gene to be knocked out – the up-rev and do-fwd primers should start further within the gene to be knocked out.
Step 2: Amplification of fragments
We use HOTstar master mix from Qiagen. For a 50 ml reaction:
25 ml master mix
2 ml of each primer (10 pmol/ ml)
1 ml of template
20 ml dH2O
primers (template):
up region: up fwd, up rev (choromosomal DNA)
down region: do fwd, do rev (chromosomal DNA)
cassette: primers and template according to primer table
PCR cycle:
15 min 95oC
20-30 sec 94oC
20-40 sec (temp. to be determined according to Tm of primers)
1-2 min 72oC (depending on length of cassette used)
10 min 72oC
Purify reaction using PCR purification kit (Qiagen) – elute in 30-35 ml.
Run 1-5 ml on gel to determine amount.
Step 3: Joining PCR
Using HOTstar master mix from Qiagen (for a 50 ml reaction):
25 ml master mix
2 ml of each primer (10 pmol/ ml)
1-4 ml of purified up and down fragments (corresponding to 200-300 ng)
2-6 ml of purified cassette fragment (300-500 ng)
dH2O – adjust vol. to 50 ml
(It seems to work best if you keep about a 1:2 ratio between flanking region:cassette)
Primers:
up-fwd, do-rev
PCR Cycle
15 min 95oC
20-30 sec 94oC
20-40 sec (temp. to be determined according to Tm of primers)
3-5 min 72oC (depending on length of cassette, up and down used)
20-30 sec 94oC
20-40 sec (temp. to be determined according to Tm of primers)
3-5 min + 20 sec (each round) 72oC
(depending on length of cassette, up and down used)
10 min 72oC
Run ~ 5 ml of reactions out on gel, purify remaining 45 ml using PCR purification kit – elute in 30-35 ml.
Step 4: Transformation:
To 0.5-1 ml competent cells add 5-10 ml of purified product. Follow your Bacillus transformation procedure.
Spin down cells, discard most of the supernatant (leave about 100-150 ml ), plate on LB+antibiotic plates. Incubate at 37oC.
Step 5: Screen colonies for verification (using colony-PCR)
for a 20 ml reaction:
10 ml master mix
1 ml of each primer (10 pmol/ ml)
8 ml dH2O
Primers: up fwd primer + proper check rev primer (according to table)
template: add cells (not too much!!) directly from colony to be screened
PCR cycle:
15 min 95oC
20-30 sec 94oC
20-40 sec (temp. to be determined according to Tm of primers)
1-2 min 72oC
10 min 72oC
run on gel - should give about 1 kb fragment in positive clones and no product in negative control.
TIPS:
· When using the Cat or Tet cassette the colonies seem to take about 2 days to come up
· We don’t usually use 2 rounds of joining PCR (one without primers), as described in the original protocol. However, if you are unable to get product with one round 2 rounds might work.
· Increasing the amount of cassette template might help if you don’t get joining product.
Reference:
A Wach (1996): PCR-synthesis of marker cassettes with long-flanking homology regions for disruptions in S. cerevisiae, Yeast 12: 259-265