pcr product - hello all (Jun/29/2010 )

i was extracted RNA from goose then i converted it to first stand cDNA and then i used house keeping gene (18s and b actine)and other degenerative primer but i couldn't find any bands even with the house keeping gene, can any one help me in this problem

wael on Jun 29 2010, 07:35 PM said:

Change the annealing temperature by a factor of one. Either increse or decrease. How are you fixing your annealing temperature. In some cases the formulas dont work. Do a simple calculation. From the data sheet obtained from the manufacturer, add the forward and reverse primer and divide by 2 and substract 2 from that. Generally that will work for the annealing. Incase it is not, increase or decrease the factor by one. If you are near ur annealing temperature, u should atlease see the house keeping gene. When there are no bands, it means that that the exp as such is wrong. Be careful when adding ur reagents. Hope this will be of some help.

jaya