Hello,
I have two cells lines: A375 and Mel-ST. This last one is an immortalized cell line from primary melanocytes. The BRAF gene should be mutated with V600E mutation in A375 cells but not in Mel-ST cells. I would like to check it out, but I am not an expert in sequencing. Is it ok to extract the DNA with Qiagen DNeasy kit and to do a PCR to amplify BRAF? In this case, how should I design the primers for my PCR and the ones for sequencing? I guess the last ones should be more close to the SNP or anyways inside the amplified sequence. What about the lenght of the sequence I amplify? N.B. we use eurofins service for sequencing.
Hi,
For Sanger sequencing you can have a maximum read size of ~800bp. It is better to design primers such that the SNP you study is somewhere near the centre. For designing PCR primers you can use NCBI PRIMER-BLAST (a very easy tool to check amplicon size and specificity). Regarding the DNA extraction you can try manual method or by the kits. The primers for PCR and the sequencing are generally the same.
Good luck.
In addition to Murthy's response, it is important that your PCR product when resolved on gel, gives only one band. Also ensure your samples are submitted for sequencing as soon as possible after PCR and purification.
Ciao Martina,
As both Murthy and Deborah said you should consider that the primers should include the SNP somehow in center.
Use any bio-informatics tools free available on internet to design them, Again NCBI PRIMER BLAST could be a good and simple choice.
The lenght of the sequence you'll get depends by your primers location which will also determine the lenght of the final PCR product.
I also reccomend you to check prior to sequencing the PCR product on agarose gel electrophoresis to be sure that is a clean, single band, good amplicon so you do not get any troubles in sequencing later one.
You check the result of DNA extraction by readings at spectrophotometer or by gel electrophoresis and then proceed with the PCR.
Any other help let me know.
Best of luck,
Stefania
Thank to all for the helpful suggestions. Since my exon is about 100bp, I was just wondering if there was a lower limit for the amplicon lenght. Is it ok to amplify a fragment between 100 and 200 bp?
I suggest you design your primers to amplify a fragment between 200-300bp and don't let your SNP position be close to the ends, middle should be fine.
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