Since i only measure the conc of RNA, how do i know how much of RNA should i put so that the product cDNA is in the range of acceptable template conc?
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#2
Trof
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Posted 23 February 2011 - 10:12 AM
We just put the same amount of RNA to RT reaction, usualy 1 ug (and use 2ul of 20ul reaction as a template). But important thing is abundance of your target gene(s), that may be too much or too little, you can't tell unless you try that.
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RecklessTech
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Posted 23 February 2011 - 12:12 PM
We pretty much go with a 100% conversion efficiency for RT. If you put 1 ug/ul of RNA in, you'll get 1 ug/ul of cDNA. It's not exact but it works. 1ug is usually more than enough.
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hianghao
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Posted 24 February 2011 - 12:04 AM
Trof, on 23 February 2011 - 10:12 AM, said:
We just put the same amount of RNA to RT reaction, usualy 1 ug (and use 2ul of 20ul reaction as a template). But important thing is abundance of your target gene(s), that may be too much or too little, you can't tell unless you try that.
Meaning use 1ug of RNA for RT in 20ul, then 2ul of cDNA from the rxn?
My extractions yield very low conc of RNA: 20-40 ug/ml. The max amount of RNA that i can use for a 25ul rxn is only 0.2ug.
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Posted 27 February 2011 - 12:19 AM
hianghao, on 24 February 2011 - 12:04 AM, said:
Meaning use 1ug of RNA for RT in 20ul, then 2ul of cDNA from the rxn?
My extractions yield very low conc of RNA: 20-40 ug/ml. The max amount of RNA that i can use for a 25ul rxn is only 0.2ug.
My extractions yield very low conc of RNA: 20-40 ug/ml. The max amount of RNA that i can use for a 25ul rxn is only 0.2ug.
Well then you have to stick with 200ng (it could be enough, as I said, depends on the gene) or try other ways how to increase RNA yield. Or use TaqMan PreAmp Master Mix from ABI, to pre-amplificate your cDNA (but I have no experience with it).
Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.
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#6
hianghao
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Posted 28 February 2011 - 03:05 AM
Trof, on 27 February 2011 - 12:19 AM, said:
hianghao, on 24 February 2011 - 12:04 AM, said:
Meaning use 1ug of RNA for RT in 20ul, then 2ul of cDNA from the rxn?
My extractions yield very low conc of RNA: 20-40 ug/ml. The max amount of RNA that i can use for a 25ul rxn is only 0.2ug.
My extractions yield very low conc of RNA: 20-40 ug/ml. The max amount of RNA that i can use for a 25ul rxn is only 0.2ug.
Well then you have to stick with 200ng (it could be enough, as I said, depends on the gene) or try other ways how to increase RNA yield. Or use TaqMan PreAmp Master Mix from ABI, to pre-amplificate your cDNA (but I have no experience with it).
I see! Thanks for the information!
