Help with biotin-labeled DNA probes

carlos h. pedemonte CPedemonte at UH.EDU
Thu Nov 17 11:05:06 EST 1994


Hi:

We are using the BioPrime DNA Labeling System from Gibco to produce
biotinylated probes. This kit has biotinylated dCTP.  We think that the
biotinylated probe has been produced because we have dot blotted a small
amount on an identical membrane and detected the presence with extravidin-
peroxidase and ECL.  However, we cannot detect any of the G3PDH mRNA
species after reacting the synthesized probe with the RNA transferred from
a gel by capillary electrophoresis following an overnight hybridization. We
have used all the conditions described in the Gibco protocol to synthesize
the probe.  In addition, we have tried some alternatives (i.e. low
stringency).  Using Gibco's Random Primers DNA Labeling System and P-32
labeled dCTP, we are able to detect the G3PDH mRNA species in as little as
one hour using identical hybridization conditions.

1)  We think that one problem can be that the presence of the biotin
molecules greatly reduced the affinity of the probe for the RNA. Is this
possible?
2) Has somebody used this kit and found that the conditions described in
the protocol (by Gibco) are not the best ones?
3) After the probe is prepared, is it necessary to clean the probe or is it
possible to use it without cleaning.
4) Does the type of membrane used to fix the target (i.e. RNA) make a
difference when using a biotinylated probe?

We will greatly appreciate any suggestion and comments. Thank you,

carlos and mike

______________________________________________________________________________
carlos h. pedemonte             Phone:   713-743-1228
University of Houston                    713-743-1211
Dept. of Pharmacology           Fax:     713-743-1229
Houston, TX 77459-5515          e-mail: pedemonte at jetson.uh.edu





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