DNA stuck in wells
smori at nmsu.edu
Wed Jan 11 16:41:06 EST 1995
Paul N Hengen (pnh at fcsparc6.ncifcrf.gov) wrote:
: In article <3enkg7$68i at dns1.NMSU.Edu> smori at nmsu.edu (Shahram Mori) writes:
: >: I've tried the 4% non-deturing gel (a band-shift assay) with TBE and TAE,
: >: with Scholler buffer and BCA and the probe (144bp) doesn't seem to move
: >: any distance into the gel even in the lane with just buffer and probe.
: > Do you heat up your DNA prior to loading?
: > It maybe that 2ndary structures might prevent it from entering. I boil for
: > 2min and place on ice immediately -> load.
: I don't recommend this because it will certainly kill the gel-shift experiment.
: The shift assay is based on protein binding to DNA causing a change in mobility.
: Since the DNA in buffer looks the same, I suggest changing buffer conditions.
: The DNA in buffer should give a nice tight band on the gel.
: P.S. What is "Scholler buffer" anyway?
Of course it would be detrimental to do it in a non-denaturing GMSA. I only
boil mine for running denaturing PAGE and agarose gels. In these cases, it is
important to heat your DNA to make it single stranded and linear to allow for
the DNA to enter the gel.
I hope I did not confuse you with my answer.
Shahram Mori _/\_
Program in Molecular Biology _\ /_
Dept. of chemistry and Biochemistry Box 3C \_ _/
NMSU Las Cruces NM ||
More information about the Methods