Thread: gene DAB1

Hi!!!
I would like to know the exact aminoacid it codifies this human gene, because in some papers they say that it codifies 555 and others say 588.
On the other hand, I am trying to amplify this gene by PCR technique with the very good enzyme that is the FAast Start but even that, it has no results... if someone has made a PCR of this gene I would ask to help the condicion of the reaction...o maybe in the celular line Im trying on is not the best (HEK, HELA and JK cells)
please help!

Hi!!!
I would like to know the exact aminoacid it codifies this human gene, because in some papers they say that it codifies 555 and others say 588.
On the other hand, I am trying to amplify this gene by PCR technique with the very good enzyme that is the FAast Start but even that, it has no results... if someone has made a PCR of this gene I would ask to help the condicion of the reaction...o maybe in the celular line Im trying on is not the best (HEK, HELA and JK cells)
please help!

Dear Kitty,
Hm after a little look on NCBI, it seems to encode 555 amino acids:

http://www.ncbi.nlm.nih.gov/CCDS/Ccd...DATA=CCDS607.1

...however this may vary if there are different isoforms.

Have you tried using fresh reagents?

Are you sure that the primers are optimal?

Have you checked the recommended conditions when using the Fast Start Taq polymerase? (greater than equal to 2 minutes at 95*C denaturation step; 72*C elongation; Mg2+ dependency; dNTPs at 200umol/l for each dNTP).

Apparently, Tween20, DMSO, gelatine, glycerol, betaine, spermidine, BSA, and ammonium sulfate additives have all been reported to have enhancing effects:

https://www.roche-applied-science.co...1&countryId=uk

Why are HEK cells not a suitable choice of cell line? Presumably you are trying to transfect something in (please correct if wrong); such a cell line is apparently fairly easy to transfect?

Your choice of cell line will ultimately depend upon what you want to find out.

Best wishes,

Tridimity
:P