I have extracted the genomic DNA and purified it by using CTAB method. I would like to amplify my genomic DNA by using PCR method.Can anybody share which PCR method do I need to used since Ive tried many time on my PCR method but I did not get any result from sequencing. the sequencing result shows something else (eg:bacterium) and not what I want to (eg:farnesyl synthase)
Zahidah, We need more details to provide a proper answer on this, like source of sample, what gene you want to amplify, etc.
If you know your target sequence you have to design the primer first.
Well, Kaushik Bhattacharjee is absolutely right.
Your DNA sample might contain DNA of your target organism and other organisms too, if the target organism did not grew sterile. But that does not mater, because your PCR should be sequence (and therefor organism) specific by using well designed primers.
Getting a sequencing result implies you got a single band in PCR, right? Well it might be not your target sequence. Where did you get your primers?
btw: title did not match with question. topic: DNA isolation problem, question: PCR problem.
From readings, i found that this enzyme is universal in bacteria (a phylogenetic marker in phototrophic) and hence impurity in DNA can cause matching of your DNA sequence to bacteria origin.
Solution: Compare the DNA seq. from both organisms (your target vs bact.) and redesign primers that align well to your target.
thank you for all your feedback. i really appreatiate.btw ive used the primer that i designed before to amplify my cDNA of that gene and it succeeded. but for gDNA amplification, the gel result consist of a few bands with different size and i have taken all the bands to be sequenced. so what should i do in order to amplifiy the correct target?
May try to (if possible):
1. Purify DNA of your interest
2. Adjust anneling temp (0.5c below primer tm) or/and extension time according to expected size of ur target amplicon (1kb/min)
It is not clear from the question what was the starting material. The method of choice in getting good genomic DNA depends on what kind of material is used as the source of DNA. If the target sequence is relatively small e.g. microsatellite sequence then chelex works best. For sequencing reactions organic method though time and labor intensive gives good quality gDNA. You can also try any of the commercial kits developed by Qiagen.
it is probably too late to be useful but if your primers work on cdna then they will work on many similar genes from many species but if your primers were intronic then their sequences should be unique to the organism that you are looking at even in the presence of the same gene from other species
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