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 37C

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:

94C for 5 minutes

94C for 1 minute

60C for 1 minute 30 cycles

72C for 1 minute

72C for 10 minutes

4C 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 37C for 2 hours

4. Run on 2% low-melt gel

5. Excise 1.6 kb band