Probe DNA Preparation for Southern Blotting

 

IRS-1 Probe DNA Prep

����� 1.�� From glycerol stocks (in �80�C 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 �20�C 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 37�C.

����� 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 45-55�C 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:

����������� 94�C for 5 minutes

����������� 94�C for 1 minute

����������� 60�C for 1 minute����� ����� 30 cycles

����������� 72�C for 1 minute

����������� 72�C for 10 minutes

����������� 4�C 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 45-55�C 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 �20�C 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 37�C.

����� 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 55�C 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 45-55�C 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