SOUTHERN PROBE DNA PREPARATION
IRS-1
1. Grow
up bacteria (use amp LB) from glycerol stocks
2. Use
a Qiagen prep to purify the DNA
3. Digest
plasmid DNA as follows:
25 ml DNA
2.5 ml Xba1
2.5 ml BamH1
3.0 ml Buffer 4
0.5 ml BSA
4. Digest
for 2 hours at 37ºC
5. Run
digest on a 2% low-melt gel
6. Excise
the 300 b.p. band
7. Use
Mermaid kit to clean (use ~ 10 ml GlassFOG)
8. Re-suspend
in ~20 ml of sterile water
IRS-2
1. Prepare
PCR reaction:
5 ml buffer
1 ml dNTP mix
2 ml 3’ primer (with green tape)
2 ml 5’ primer (with green tape)
0.5 ml Taq
2 ml IRS-2 probe DNA
37.5 ml sterile water
2. PCR
profile:
94ºC for 5 minutes
94ºC for 1 minute
60ºC for 1 minute 30 cycles
72ºC for 1 minute
72ºC for 10 minutes
4ºC for 1 hour
3.
STF
1. Grow
up bacteria and purify DNA via Qiagen prep
2. Digest
as follows:
2.5 ml Xba1
2.5 ml EcoR1
5 ml buffer 2
0.5 ml BSA
25 ml DNA
3. Run
digest on 2% low-melt gel.
4. Excise
which band?
5. Purify
with GeneClean or Mermaid kit?
IGF
1. Digest
plasmid with BamH1 and HincII
2. Excise
460 b.p. band
IMM
1. Plasmid
is RSVTEK
2. Digest
as follows:
30 ml DNA
10 ml Pst1
10 ml buffer 3
50 ml sterile water
3. Digest
at 37ºC for 2 hours
4. Run
on 2% low-melt gel
5. Excise
1.6 kb band