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Southern - Southern Blot Digestion (Oct/12/2005 )

We haven't succeeded with our Southern for 3 times in a row. The control looks good, so it doesn't appear to be a probe issue. However, the digest doesn't look like a full smear (smears from the well only about halfway down the gel). We use concentrated Bam, 8ul for a 100ul digestion (need this volume to digest 15 ug of gDNA). Digests run overnight at 37 degrees, but don't appear to fully digest the gDNA (control digest looks good). The purity is greater than 1.7 260/280.

Should I use a different protocol to get more concentrated gDNA (usually is around 300 ng/ul)?

What suggestions do you have for a full digest of the genomic DNA? blink.gif

-UTSWSteve-

QUOTE (UTSWSteve @ Oct 13 2005, 12:47 AM)
We haven't succeeded with our Southern for 3 times in a row. The control looks good, so it doesn't appear to be a probe issue. However, the digest doesn't look like a full smear (smears from the well only about halfway down the gel). We use concentrated Bam, 8ul for a 100ul digestion (need this volume to digest 15 ug of gDNA). Digests run overnight at 37 degrees, but don't appear to fully digest the gDNA (control digest looks good). The purity is greater than 1.7 260/280.

Should I use a different protocol to get more concentrated gDNA (usually is around 300 ng/ul)?

What suggestions do you have for a full digest of the genomic DNA? blink.gif



Hi !
I am also facing same problems as u ..
can u please let me know as what "control DNA" digestion did u use ?

meeta sad.gif

-meeta-

QUOTE (meeta @ Oct 13 2005, 11:37 AM)
QUOTE (UTSWSteve @ Oct 13 2005, 12:47 AM)
We haven't succeeded with our Southern for 3 times in a row. The control looks good, so it doesn't appear to be a probe issue. However, the digest doesn't look like a full smear (smears from the well only about halfway down the gel). We use concentrated Bam, 8ul for a 100ul digestion (need this volume to digest 15 ug of gDNA). Digests run overnight at 37 degrees, but don't appear to fully digest the gDNA (control digest looks good). The purity is greater than 1.7 260/280.

Should I use a different protocol to get more concentrated gDNA (usually is around 300 ng/ul)?

What suggestions do you have for a full digest of the genomic DNA? blink.gif



Hi !
I am also facing same problems as u ..
can u please let me know as what "control DNA" digestion did u use ?

meeta sad.gif



hi
maybe this comes very late
for complete digestion of genomic dna from plant
u have to dilute the dna in DW and use high concentration of enzyme arond 10-15 units
digest it o/n
also the company of the enzyme will also afect , for example i used TAKARA pstI , there was no digestion but MBI fermentas enzymes worked fine .

for digestion

for example
if you use 100 microgram dna -200 microgram
Use 10X enzyme buffer- 50 microliter ( for final reaction volume 500 microliter)
enzyme like EcoRI or hindIII are better for digestion unless ur looking for ur transgene and this enzyme cuts inside ur gene. then use the enzymes that cut at either side of ur gene--- 10U -usually 5 microliter

DW - make up to 495 microliter ( if using 5 microliter of enzyme )

mix well and pulse and keep at the right temp

after one hour again add an extra 10U thats 5 microliter mix and pulse

keep in app temp Over night

next day u will get fully digested DNA


hope its not too late
regards
laxmi

-phytoviridae-

I have one question.
If you have big volume(500ul) of digested DNA, do you usually precipitate again for gel electrophoresis?
Could you explain how to do it?

QUOTE (phytoviridae @ Dec 17 2007, 01:17 AM)
QUOTE (meeta @ Oct 13 2005, 11:37 AM)
QUOTE (UTSWSteve @ Oct 13 2005, 12:47 AM)
We haven't succeeded with our Southern for 3 times in a row. The control looks good, so it doesn't appear to be a probe issue. However, the digest doesn't look like a full smear (smears from the well only about halfway down the gel). We use concentrated Bam, 8ul for a 100ul digestion (need this volume to digest 15 ug of gDNA). Digests run overnight at 37 degrees, but don't appear to fully digest the gDNA (control digest looks good). The purity is greater than 1.7 260/280.

Should I use a different protocol to get more concentrated gDNA (usually is around 300 ng/ul)?

What suggestions do you have for a full digest of the genomic DNA? blink.gif



Hi !
I am also facing same problems as u ..
can u please let me know as what "control DNA" digestion did u use ?

meeta sad.gif



hi
maybe this comes very late
for complete digestion of genomic dna from plant
u have to dilute the dna in DW and use high concentration of enzyme arond 10-15 units
digest it o/n
also the company of the enzyme will also afect , for example i used TAKARA pstI , there was no digestion but MBI fermentas enzymes worked fine .

for digestion

for example
if you use 100 microgram dna -200 microgram
Use 10X enzyme buffer- 50 microliter ( for final reaction volume 500 microliter)
enzyme like EcoRI or hindIII are better for digestion unless ur looking for ur transgene and this enzyme cuts inside ur gene. then use the enzymes that cut at either side of ur gene--- 10U -usually 5 microliter

DW - make up to 495 microliter ( if using 5 microliter of enzyme )

mix well and pulse and keep at the right temp

after one hour again add an extra 10U thats 5 microliter mix and pulse

keep in app temp Over night

next day u will get fully digested DNA


hope its not too late
regards
laxmi

-MicroChipper-

hi

you can either use the full digested DNA If u have such a big gel, or concentrate using chloroform :isoamy alcohol method. better check if these chemicals has any effect on ur plant dna , or if adding any other reagent like phenol makes it better purified.

u can use chloroform: isoamy alcohol (24:1) to the DNA .Take same vol as that of the sample.
mix well by inverting( no vortexing the plant dna , it might break)
centrifuge
collect aqueous phase
then use double volume of 100 % ethanol ( ice cold) , mix by inverting and keep at -20 degree Celsius for one hour
centrifuge
wash with 70% alcohol
centrifuge(If u want u can repeat the step)
air dry
and dissolve in TE or water( usually lesser amount than original volume, like if you had 500 use 100 micro liter in the last step , u will get higher conc digested DNA) . overnight at 4 degree ( best)
and then use it
Its usually better to purifiy it after digestion, though depends on ur specimen.

u will get the variation of this protocol in the net and other tips too try searching

all the best

-phytoviridae-