I am now working with qPCR for quantifying the gene copies of each genes from a pooled culture of yeast.
In designing the primers for each gene for qPCR.
I'm searching for the answers of the following questions:
1. Which method or kits do you recommend for the genomic DNA preparation from yeast?
2. What is the optimal size of the PCR product for qPCR analysis? (Is there a problems in case the size is about 70 bp.
3. If the answer is No, what is the smallest size (>bp) to ensure good qPCR quality?
4. If there are the differences of Tm among the primers for each gene, is it OK to perform in a single PCR panel at the same annealing temp?
5. If No, what are your recommendations?
6. If the product size and primer sequences are all different between the samples, I think that the signal intensity, profiles and Ct are possibly very different between samples. In this case, are there any methods for determining the Ct values that represent correct gene copy numbers?
For extracting DNA from yeast, I have used the MoBio Ultraclean microbial DNA isolation kit:
http://www.mobio.com/microbial-dna-isolation/ultraclean-microbial-dna-isolation-kit.html
For qPCR, there is no problem with a product of 70 bp. I have used products as small as 50 bp for Sybr green based detection. For TaqMan assays, the minimum size is dictated by the need to include a probe sequence within the amplicon.
Calibration is a difficult issue, since the qPCR assays may vary in efficiency and, if you are using Sybr-green, the size/dye-binding capacity of each different amplicon may be different--so you are correct that Ct values for different qPCR assays can not be compared directly. You may need to isolate pure gene fragments of the segments you are interested in, carefully quantitate them by UV absorption and prepare calibration curves for each qPCR assay. If you do not need absolute quantitation, but only need to study how copy numbers change in each sample, then you may be able to identify a "reference sequence" which has the same copy number in each sample and use that to calculate relative differences.
Dear Dong-Myung,
In my lab we are studying yeast genetocs since some 30 years now. WE began at a time there was NO a single kit to prepare genomic DNA.
We are still not using kit but the lab protocol which gives the best results, especially for PCR amplifications.
Please find this protocol as a joined pdf file to this answer.
Best wishes,
Prof Philippe Urban
Dear Philippe,
thanks a lot for your answers and the protocol.
In addition,
is there any commercial kits for the perparation of genomic DNA from yeast that can provide the good results for qPCR?
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