Posted 16 March 2005 - 02:41 PM
I am repeating my PCR now. Acturally I already got my PCR product at 62C degree. But now when I repeat it,I can't get my products again. I only get the smeared band from top to the bottom of the gel. the smeared band is very bright.I don't know what's the problem of my PCR.could you give me some suggestions?
thanks a lot
Posted 16 March 2005 - 06:17 PM
Be careful on how you hold the reagent tubes between your fingers.
DO NOT trust sterilized water from someone else. Make your own the day before.
PCR sounds like something trivial. It is not.
Posted 16 March 2005 - 08:17 PM
Do you quantitate your template prio PCR? When yoy have smeared band from top to the bottom of the gel most likely due to the Excessive omount of templete.
When you carry out DNA quantification be aware of you DNA purity. The ratio of A260:A280 has to be > 1.5 and you anly put < 1ug per 50ul reaction.
Try it out! Good luck!
Posted 24 March 2005 - 11:34 AM
after checking the DNA ,,,u can make several test tubes using the same annealing temperaure u r on
change the source of the components u r using ....in one tube change ur Taq ,,,in another one use a new amount of ur working primers
in the other change ur dNPS
sometimes one component is jus no working well
Edited by lula, 24 March 2005 - 11:35 AM.
Posted 24 March 2005 - 12:23 PM
Posted 26 March 2005 - 01:50 AM
What MoleculeMan say is very true. What extraction method are you using?
Try to chech ratio of A260:A280.