SOUTHERN BLOT:
I. Draft cDNA gel onto saran wrap prior to denaturation, neutralization & transfer system.
II. Denaturation of DNA for 45 mins. in 250 to 300 ml per tray of gels. (Need to keep gels
submerged in solution-press down gel if it surfaces to the top)
0.4 M NaoH: 1M= 58.44g/l, therefore 0.4M=23.4g/l
1M Nacl : 1M=40g/l
w/ gentle agitation
III.Rinse gels with ddH2O
IV. Neutralization of gel 2x/ 20 minutes each.
1M Tris: Trizma Hcl 157.6 g/l,pH 7.4
1M Nacl: 1M=40g/l
w/ gentle agitation
V.Transfer system:
-glass or plexiglass tray or dish
-20xssc or 20xsspe (use stronger ionic strength solution for smaller DNA(<500
nucleotides) fragments. Nylon membrane will bind small DNA fragments better than
nitrocellulose.
-use a 1000ul blue tip rack
-place a gel tray over blue tip rack
-long wet whatman paper, place over gel tray & hangs down into the transfer solution
-place agarose gel backside up so samples are on top, cut out the right corner of gel for
marking
-cut out a piece of nylon filter of equal size to the gel.Soak nylon filter in transfer buffer
for 5 mins. Then place it over agarose gel. Press out bubbles with glass rod. Cut out right
corner of nylon filter so after transfer there will be a cut on the left side of right side of
paper. Use a #2 paper to trace the upper wells of the agarose gel.
-Cover nylon filter area with two wet cut to size whatman paper, press out bubbles.
-Overlay entire setup with saran wrap & cut away the part covering the gel+nylon paper+
whatman paper or the area over the blue tip rack.
-Stack paper towels on top of saran wrap free area, place a plate over paper towels &
weigh it down with a heavy object.
-Transfer for 8-24 hours.
VI. Cross-linking transfered DNA on nylon filter:
-use stratalinker auto-link the correct side of nylon filter where a cut is at the left side of
paper while you are facing it. Store nylon filter in saran wrap in clean & dry area until prehybib.
VII. Prepare pre-hybridizing solution & pre-hybridize nylon filter:
-pre-hybridizing solution for a RNA:
6x SSC or 6x SSPE
5x Denhardt’s reagent
0.5% SDS
100ug/ml denatured, fragmented salmon sperm DNA
to make up 10 ml:
3 ml 20x SSC or 20x SSPE
1 ml 5x Denhardt’s reagent
0.5ml 10% SDS
100ug/ml salmon sperm DNA
-prehybridize nylon filter in prehyb. Solution for 1-2 hrs.
Pg.2 Southern blot cont’d
in 52C hybridization oven.
-For oligonucleotide hybridization buffer 100ml:
-from 20xSSC to make up 5x of 25 ml
-.2M NaH2PO4 to make up .02 M of 10 ml
-20xSDS to make up 7x of 35 ml
-50x denhardt to make up 10x of 20 ml
-10mg/ml SSDNA to mak up 100ug/ml of 1ul
-ddH2O 13 ml to make up total volume of 100ml
VIII. Prepare prob:
for single-cell:
-DNA template (2ul from aRNA)
-10x TSC buffer (2.5ul)
-3 rNTP’s mix 2.5mM (2.5ul)
-DTT 100mM (2ul)
-32P-CTP (3ul)
-RNAsin (0.5ul)
-dH2O (up to 21ul)
-T7 RNA polymerase 1000U/ul (1ul)
incubate for 4 hrs. In 37C. Heat inactivate for 3mins. In 95 C prior to adding directly into
chamber. Hybridize nylon filter & radioactive probe for <48 hrs. In 52C oven.
for oliogonucleotide:
-oligonucleotide 2.5pmole (2ul)
-10x reaction buffer (5ul)
-T4 Kinase 4U (1ul)
-[32P] gamma-dATP (5ul)
-ddH2O (47 ul)
incubate probe mixture for 30 mins. In 37C. Add probe directly into prehyb. Chamber
incubate for 24 hrs.
IX. Wash :
for single cell 2nd round aRNA:
-solution I: 2x SSC + 0.1% SDS, wash 2x in RT for 30 mins/wash
-solution II: .1X SSC + 0.5% SDS, Wash for 45 mins. In 37C
for oligonucleotide:
-2ml of 20x SSC
-0.1 ml of 20% SDS
-18 ml of ddH2O
wash for 15 mins. Each 2x
cover blot with saran wrap & ready for exposure to x-ray film.
X. Exposure to x-ray film:
-sandwich wrapped blot between film & intensifying screen
-expose in -80C for O/N to 2 days depend upon activity
-nylon filter can be reused or relabelled with other probes