I am working on Phaseolus vulgaris,(common beans) and extracted DNA from leaves samples. I am targeting the phaseolin gene and have utilized phaseolin specific pair of primers, using both hotstart PCR premix Biooneer company and Go taq Promega.
The primers however worked well during optimization, but are failing to amplify completely now that am dong PCR. Any recommendations will be appreciated.
There is not enough information to answer this question. A picture of the gel would help but in the most general terms a pcr can fail with poor quality,toomuch or too little dna. Measure the OD260/280 to check the quality of the dna and also to measure the amount of dna as too much dna can be inhibitary to pcr. Low quality ( od 260/280 much below 1.8) also has difficulty melting so does not amplify well and also make it hard to measure how much dna you are adding as some of it is protein also absorbing at 260nM
I think that you need to first check the concentration of DNA because, you can have DNA in the reaction but it is not enough for the reaction, check the ratio, it could also be due to the parameters that you set the run, you need to check and optimize as well, or even due to the reagents you are using.
Thanks
Gadji_M
My suggestion is for you to think about the possibility of contaminants in this DNA sample. Secondly, Check the purity as well as the concentration of the DNA sample. Apart from that, the annealing temperature plays a key role in PCR .
Thank you for your responses@paul Randut@Swagat @Mahamat. This picture attached here is how the gel electrophoretogram appeared,for subsequent tests. However the previous tests show nothing at all on the gel. What is the best way to remove contaminants from an already extracted sample?
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