Hi everyone! Attached you will a EtBR gel showing failed PCR results in lanes 2 and 3, with positive results for a DNA ladder in lane 1 (100 bases). Additionally, I have attached a photo of the reactant mixtures used for this PCR protocol. My question is: Do bear and/or squirrel actin primers work in our new mammalian model system in order to produce hybridization probes for actin mRNA? So, I am only concerned with seeing a result, not quantifying the result.
I understand how to calculate concentrations of all reactants, but I do not know where to begin with optimization of my protocol.
Hi,
Since you are seeing no amplification at the moment, you should try conditions with lower stringency to get ANY amplifications.
To do so, I would suggest you to try:
Once you get amplifications (I assume you would have more than one products), you can gradually reverse these parameters to look for the optimal condition.
Also, is it possible to include the positive control (e.g., cloned cDNA) for the PCR reaction?
By the way, I'm just curious. Is it anything to do with hibernation, judging from bear and squirrel?
Hi Jared
Besides the very relevant comments already posted, I would advice you to put some DMSO in your reaction mixture. You can start by 6% and go as higher as 12%. This additive decreases stringency and it works nicely when you are amplifying sequences with high GC content.
Best
Paco
hi
u should:
1. check ur primer with OligoAnalyzer 3.1---> http://eu.idtdna.com/calc/analyzer
2.use gradient PCR to obtain correct TM
3.use higher mg concentration
be succeed.
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