Probe DNA Preparation for Southern Blotting

IRS-1 Probe DNA Prep

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

      2.   Use a Qiagen prep to purify the DNA.

      3.      Perform the following digest:

            25 m l DNA (usually found in a tube labeled “IRS-1 DNA for Probe” in the �20ºC freezer)

            2.5 m l Xba1

            2.5 m l BamH1

            3.0 m l buffer 4

            0.5 m l 10X BSA

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

      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 m l of resuspended GLASSFOG per m g 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 m l High Salt Binding Solution

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

            i.    add 300 m l 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 45-55ºC 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 m l PCR reaction:

            5 m l buffer

            5 m l dNTP mix

            3 m l 3’ primer (labeled “JS-25” with green tape)

            3 m l 5’ primer (labeled “JS-26” with green tape)

            1 m l Taq

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

            31 m l sterile H₂ O

      2.   The following PCR profile works:

            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.   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 m l of resuspended GLASSFOG per m g 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 m l High Salt Binding Solution

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

            i.    add 300 m l 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 45-55ºC 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 m l 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 m l DNA (tube labeled “IGF Plasmid DNA for Probe” in the �20ºC freezer)

            2.5 m l BamH1

            2.5 m l HincII

            3.0 m l buffer 3

            0.5 m l 10X BSA

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

      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 55ºC for 1-2 minutes; mix; incubate again for 3-4 minutes

            c.   add 8 m l of resuspended GLASSFOG per m g 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 m l High Salt Binding Solution

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

            i.    add 300 m l 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 45-55ºC 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