real-time PCR

[fibonacci]

hi..

i need some help from the members who has some experience doing the real-time PCR...

i'm planning to do the one-step REALTIME-PCR and i have bought the QuantiFast SYBR Green kit, Qiagen..

i have some questions about real-time PCR before i starting the experiment.

 

the questions are :-

 

1) how to prepare the standard curve in real-time?? what template should i use to generate the standard curve?? DNA(plasmid DNA which contain my cDNA) @ RNA ??

 

2) after i designing the primer, i testing it by doing RT-PCR and i successfully amplified the genes of interest with the expected sizes. but, in the gel picture (2% agarose), i found out that there are some smearing under the expected band (means that the size is smaller than the expected sizes). does this smearing will affect when i do my real-time experiment???

 

3) in the QuantiFast SYBR Green kit protocol, it stated that the annealing/extension process were combined at 60C for 30 seconds (it also stated that this temperature should also be used for all primer sets with a Tm well below 60C). i have designing the primers with Tm around 55C-57C.. so, which Tm should i use?? 55C-57C or 60C???

 

hope someone can help me...

thanks..

[CharonY]

1) This is always the basic problem with QPCRs. The most correct standard would be your target RNA in known quantities. This is rarely feasible, though. Other possibilities are chromsomal DNA, or plasmid DNA, provided that accurate concentrations are available and that your target is in there (obviously). In both cases you should be aware that due to the missing RT you will introduce a bias. If you have no reference DNA available with known concentrations you will be limited to a semi-quantitative measurement.

2) It depends where the smear is. If the size is very small, it could be the primer cloud. It does not interfere too much. If they are indeed bands, meaning fully double strand product, then the PCR is not stringent enough. In the latter case SYBR green will also intercalate there and give more signal. But after the run one usually makes a melting analysis and you should see additional products there.

3) Start with 60. IIRC the kit is desinged to run well on that temperature. 5 degrees is generally not a problem.

[fibonacci]

thanks for the info...

i will try first...

[Benalwaleed]

http://rapidshare.com/files/247707424/rt_pcr_1step.pdf

http://rapidshare.com/files/247707851/rt_pcr_2step.pdf