Hi everyone and good morning!
In our lab we want to make a sanger sequence opf the RPGR gene (ORF1/ NM_001034853.2) but is technically very dificult to make any PCR within that zones (it´s a mutational hotspot so, it has to be made)
¿anyone has any idea? we have arrived at the point to have very few not specific bands (so we cut the band of interest and so on) but I wanted to learn from others experience working on STR´s zones.
thank you very much... it´s technically challenging and fun!
when you get to the sequencing step you will need to sequence from both ends as size polymorphisms will mean that your sequence will be messy after the change, You may be able to subtract one sequence from the other to see what the base changes are or there is software online allowing analysis of mixed sequences but you may end up cloning amplimers and sequencing clones to confirm changes
we send for other mutations both forward and reverse primers and I observed this phenomena on frameshift mutations (wich make them easy to identify on the SeqMan) I'ill make what you say and maybe send also some internal primers.
thank you Paul!!!
I also wanted to design the primers for some mutant genes of cancer, can you help me how can I design primers for the mutation analysis of genes through PCR?
I will recomend you for primer design to make them within the exon sequence if it´s possible (it´s something I make on my experience).
Then be sure to have a 50-60% content of GC with a melting temperature of +-60ºC ( I design my primers for upper temperatures but you can try and experience for yourself what works for you)
Also to have G´s or C´s on the tails, it makes them more stable and affinity like.
Don´t aim for a big Amplicon, it will make your sanger seq unconsistent, min 200bp max 800bp (most "battle" polymerases will do great).
Always perform a melting curve (try your primers in different temperatures and diferent additives, DMSO, Mgcl2 and so on..) because if you starting from zero...¿why not waste a little time having better results?
Once you have your primers, you can put the sequences on the UCSC´s "in silico" PCR and search for homologous zones.
maybe Paul has more advices but I´ve run out of them, have fun!
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