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Probe DNA Preparation for Southern Blotting

Probe DNA Preparation for Southern Blotting

 

IRS-1 Probe DNA Prep

1. From glycerol stocks (in 80C freezer), grow up bacteria. Use amp LB.

2. Use a Qiagen prep to purify the DNA.

3. Perform the following digest:

25 ml DNA (usually found in a tube labeled IRS-1 DNA for Probe in the 20C freezer)

2.5 ml Xba1

2.5 ml BamH1

3.0 ml buffer 4

0.5 ml 10X BSA

4. Digest for at least two hours at 37C.

5. Run digest on a 2% low-melt agarose gel.

6. Excise the 300 bp band.

7. Use Mermaid Kit to clean:

a. weigh the excised band (1 mg 1 ml)

b. add 3 volumes of High Salt Binding Solution to the tube

c. add 8 ml of resuspended GLASSFOG per mg of DNA in tube

d. vortex tube continuously for 10 minutes

e. centrifuge at high speed for a few seconds to pellet GLASSFOG

f. remove supernatant and save aside

g. wash pellet with 200 ml High Salt Binding Solution

h. spin for 1-2 seconds and remove remaining liquid with small bore pipet

i. add 300 ml EtOH wash and resuspend GLASSFOG pellet by vortexing

j. centrifuge briefly and discard supernatant

k. repeat steps (i) and (j) one or two more times

l. after last wash, spin tube for 1-2 s; remove remaining liquid with small bore pipet

m. elute DNA from GLASSFOG by resuspending in volume of sterile water equal to

volume of GLASSFOG used in step (c)

n. incubate at 45-55C for 5 minutes

o. centrifuge for 1 minute

p. transfer supernatant (Probe DNA) to new, labeled tube

q. repeat steps (m) through (p) once and combine the elutions

8. run on 2% low-melt gel to verify band size and strength

9. determine amount of DNA to label and write on microfuge tube

 

IRS-2 Probe DNA Prep

1. Set up the following 50 ml PCR reaction:

5 ml buffer

5 ml dNTP mix

3 ml 3 primer (labeled JS-25 with green tape)

3 ml 5 primer (labeled JS-26 with green tape)

1 ml Taq

2 ml IRS-2 probe DNA (left over from previous probe DNA or in reserve tube)

31 ml sterile H2O

2. The following PCR profile works:

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. Run entire PCR product on 2% low-melt gel at 80 V for 2.5 hours.

4. Excise 250 bp band and clean with Mermaid Kit

a. weigh the excised band (1 mg 1 ml)

b. add 3 volumes of High Salt Binding Solution to the tube

c. add 8 ml of resuspended GLASSFOG per mg of DNA in tube

d. vortex tube continuously for 10 minutes

e. centrifuge at high speed for a few seconds to pellet GLASSFOG

f. remove supernatant and save aside

g. wash pellet with 200 ml High Salt Binding Solution

h. spin for 1-2 seconds and remove remaining liquid with small bore pipet

i. add 300 ml EtOH wash and resuspend GLASSFOG pellet by vortexing

j. centrifuge briefly and discard supernatant

k. repeat steps (i) and (j) one or two more times

l. after last wash, spin tube for 1-2 s; remove remaining liquid with small bore pipet

m. elute DNA from GLASSFOG by resuspending in volume of sterile water equal to

volume of GLASSFOG used in step (c)

n. incubate at 45-55C for 5 minutes

o. centrifuge for 1 minute

p. transfer supernatant (Probe DNA) to new, labeled tube

q. repeat steps (m) through (p) once and combine the elutions

5. Run 3-6 ml of DNA on 2% low-melt gel to determine size and strength

 

 

STF Probe DNA Prep

 

 

 

IGF Probe DNA Prep

1. Perform the following digest:

25 ml DNA (tube labeled IGF Plasmid DNA for Probe in the 20C freezer)

2.5 ml BamH1

2.5 ml HincII

3.0 ml buffer 3

0.5 ml 10X BSA

2. Digest for at least two hours at 37C.

3. Run digest on a 1% agarose gel.

4. Excise the 460 bp band.

5. Use GeneClean Kit to clean.

a. weigh the excised band (1 mg 1 ml)

b. add 3 volumes of NaI to the tube

c. incubate at 55C for 1-2 minutes; mix; incubate again for 3-4 minutes

c. add 8 ml of resuspended GLASSFOG per mg of DNA in tube

d. vortex tube continuously for 10 minutes

e. centrifuge at high speed for a few seconds to pellet GLASSFOG

f. remove supernatant and save aside

g. wash pellet with 200 ml High Salt Binding Solution

h. spin for 1-2 seconds and remove remaining liquid with small bore pipet

i. add 300 ml EtOH wash and resuspend GLASSFOG pellet by vortexing

j. centrifuge briefly and discard supernatant

k. repeat steps (i) and (j) one or two more times

l. after last wash, spin tube for 1-2 s; remove remaining liquid with small bore pipet

m. elute DNA from GLASSFOG by resuspending in volume of sterile water equal to

volume of GLASSFOG used in step (c)

n. incubate at 45-55C for 5 minutes

o. centrifuge for 1 minute

p. transfer supernatant (Probe DNA) to new, labeled tube

q. repeat steps (m) through (p) once and combine the elutions

 

 

Immorto (TG) Probe DNA Prep