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