newbie17425

i am using a kit for extraction. Our lab doesn't have any clean up kit.

Thanks for guiding me on how to upload the pictures. Here are the pictures The first picture is of the NTC and the other two are of the 2 unknown samples in triplicates.

Hi, I got a PCR efficiency for all my targets and samples as 1. I did a quantitation analysis but it still calculated it as 1. I know that the R^2 value should be 0.9933 and slope should be -3.32 then by formula we get 1. But as i didn't run a standard curve, how reliable is this efficiency result? (lot of questions today). Thanks

For some reason i cannot upload a picture here. I just wanted to know what does the peak represents? And if i had secondary products/contamination/primer dimers then i would have extra peaks right?

Hello again, I have extracted RNA and i am satisfied with the purity (260/280 ratio) and the yield. What is troubling me is the very low 260/230 ratio? Therefore, i wanted to know how important is the 260/230 ratio in RNA extraction. I will use the RNA for qPCR. Yield 26-35 ng/ul and 260/280 ratio is between 1.9-2.2 If i want to re-precipitate the RNA should i use sodium acetate or Lithium chloride to do this? Thanks

Hello, I want to understand how do i analyze melt curve after my qPCR? What are the different curves? I am just aware that melt curve analysis is done to check for secondary products or primer dimer formation. I would appreciate your help in understanding the melt curve. Thanks

I never did. I extracted the RNA and checked for its purity on nanodrop. Should i again check after DNAse treatment? Also, how much of this DNAse treated RNA should i take for the cDNA synthesis. I take 2ul (conisdering the yield to be 19 ng/ul) for the cDNA synthesis and recommended range is 1pg - 5ug.

Yes, i meant after adding the DNAse treatment but yes i forgot that won't change the concentration but only the volume. How did you calculate that i have 0.95 ug? What do you mean by accurate determination after DNAse treatment?

Hi, I know this question must have been answered before but i would like to confirm it again. I am using Thermo-scientific cDNA synthesis kit and it says take 1pg - 5ug of your template for cDNA synthesis. I am having a RNA yield of 19 ng/ul (micro-dissected) brain sample i.e. the hippocampus with a total volume of 50 ul. I then treat it for DNAse. So, my total volume is not more than 55 ng/ul and i want to save some RNA for future experiments. Now, to calculate 1 ug, x ul = (1 ug)/(25ng/ul) x ul = 1000/19 x = 52.63 ul Until now i was using about 2 ul of the template to synthesis cDNA. Will it be fine with 2 ul or should i inc. the volume to 50 ul for cDNA synthesis and then proceed on with the qPCR? Although the cDNA synthesis kit gives a final volume of 20 ul after addition of the template. Thanks

Hello, I recently did a qPCR (just a trial) using a ABI stepone plus system and i am stuck with the results. I am unable to understand the following: 1) Ct Mean: I have only one Ct value for a sample but it gives me a mean Ct mean value. How? Even some samples went undetermined but it still shows a Ct mean value. Why? 2) What is meant by quantity and why is that empty? 3) What is Ct Threshold? 4) I have three Tm values. I assume that is the melting temp. of the primers. Am i correct? Why are there 3 Tm values? Please find attached a screenshot of the result Thanks

Thanks Charon for your quick response. I am looking for relative quantification. I am confused (standard curve) if i have to mix all my sample cDNAs and then make a dilution series out of it (1:1,1:4,1:16,1:64 and 1:256) or i just take one sample make a dilution series out of it. Or i just make a dilution seris of only one sample.

Hello, Could someone give me an example of how to prepare a standard curve for qPCR? I read some protocols but got confused. It would really be helpful if someone could help me with an example. Also, i read that i need to prepare a standard curve for each of my plate? Is it correct? Lastly, can we run the housekeeping (HG) genes and gene of interest (GOI) on separate plates? Eg: i have 30 genes and i want to run it as triplicates. So, either i run all the GOI on one plate or omit some and run the HG with the GOI OR i could run the HG genes on a different plate. Is it correct that i need to run my HG genes for every new sample i run? Thanks