need help

[Xiangzhen Sui]

Dear sir.

Now I will be working on the quantitative PCR using ABIPRISM 7700 and
SYBR Green PCR Master Mix. I want to ask you some question.

1.Do I need use NuSieve 4% (3:1)agarose gels for check myPCR Product
Purity? How to make up it?

2. How about reaction volume of RT and concentration of Total RNA? I
have a RT kit from Invitroge. Using this kit reaction volume of RT is
20ul  and concent of Total RNA is 5ug. But in your guidelines for RT(4-2
in protocol of SYBR Green PCR Master Mix) reaction volume of RT is
100ul  and concent of Total RNA is 2ug. I do not want to buy new kit for
RT. How can I use my cDNA? Decreate concent of  Total RNA or dilute
cDNA?

3. How to know the content of cDNA, How much cDNA should I take for one
well?

Thanks
Sui



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