Southern blot help
Posted 14 June 2010 - 10:35 AM
These are the conditions of my experiment:
overnight digestion of 2ug of DNA with 20units of enzyme in a 30ul volume reaction
All product is run on a TAE 1X 0.8% agarose gel for 4hrs at 70V over ice (I'm not using EtBR anymore)
Gel is transferred overnight using capillary method with 20X SSC
Next day the membrane is air dry and fixed using UV crosslinker
membrane is prehybed for 3 hours with DIG easy hyb
and then hybridized with new easy hyb with the denatured probed
Can anyone help me with this ordeal?
Posted 14 June 2010 - 01:28 PM
You can optimize your binding and visualization with dot blots rather than doing gels. Dilute serially your dna (cut or uncut) and dot it on a membrane, crosslink, and detect to determine your limiting detection and to optimize detection conditions. Only then run gels and blot.
Posted 20 June 2010 - 11:58 AM
I am trying to perform a Southern blot of mouse genomic DNA extracted from ES cells.
This is my protocol
1. Digest 7micrograms with my restriction enzyme (5U per microgram) o/n
2. Run a 0,7% agarose gel in TAE buffer, with gel red. 45V for 24h.
3. View the smear on Image master.
4. Denaturation (0,5M NaOH + 1,5 NaCl) 2 x 20min (the fragments that I want to see have 7kb and 9kb, so I donīt do depurination)
5. Neutralization (Tris-CL 0,5M + NaCl 1,5M, pH 7,5)
6. Transfer in a vaccum device using neutral transfer buffer (20x SSC) for 1h, to Hybond N+ membrane.
7. Cross link with UV
8. Pre-hib at 55C for 1h
9. Hib with myDNA probe (430bp) labeled with alkaline phosphatase (Alkphos direct labelling and detection kit from GE - Amersham) at 55C o/n.
10. Add substrate and wait 5h until photograpg the blot on storm.
I am having huge trouble! I only see the DNA smear from my digests! I though that It was from gel red, but I already discarded that hypothesis...I can't see a specific band. Already waited 24h for revelation and the smear just intensified!
Please help me!
Posted 29 June 2010 - 07:25 PM
Posted 30 June 2010 - 05:10 AM
Posted 16 November 2010 - 01:10 PM