Hi every body, i have to tried to design a groups of primers to my work about detection of some genes in bacterium (brucella spp.) i used primer3 oline software to design of my... Show moreHi every body, i have to tried to design a groups of primers to my work about detection of some genes in bacterium (brucella spp.) i used primer3 oline software to design of my primers, list of my primers with their products:
Primer Sequence (5’- 3’) Product size
CBG F GAATTCGCCAATGAGGAAAA 575bp
R ACGATATCGGATGCGAAAAG
mviN F GCAGATCAACCTGCTCATCA 344bp
R GGCCATAGATCGCCAGAATA
manA F TCGATCCAGAAACCCAGTTC 271bp
R CATACACCACGATCCACTGC
manB F GGCTGGTTCGAGAATATCCA 228bp
R CAATCGCATACCCTGGTCTT
wbkA F GAGCGCTTAGGAATGCTGAT 309bp
R CTCCTAGGTTCCAGCCCTTT
omp25 F CGTACCTCACGGCTGGTATT 188bp
R CGTACCGGCCAGATCATAGT
omp31 F GCTGCTCCTGTTGACACCTT 257bp
R GCTGAAATCGAACCCGTAAC
znuA F CTGGGTCCGAGCATGTTTAT 465bp
R AGGCATCGAGTTTTTCTCCA
my question: 1- can i use all these primers in as multiples in one pcr reaction but i have only standrd 2X pcr master mix (promega).....12.5ul per one reaction?
2- are you know what is the best pcr thermocycler condition per all primers or per each primer?
3- can i use multiplex pcr by use two primers only?
4- what is the type of pcr thermocycler can be used in this reaction....normal or gradient pcr???
You can try a multiplex, but it will cost you a lot of optimization. All primer pairs will need to work at the same temperature, so start with the Tm that Primer3 gave you. It will be much easier and quicker to optimize the reaction in a gradient cycler, however after you have pinned the correct conditions you can proceed to using a regular cycler (keep in mind though that changing a machine might affect your reaction again, so it's better to optimize and later work on a the same machine).
You might also need to play with the Mg ions concentration, so just the standard mix might not work in this case. You should also check if the primers form different pairs do not create heterodimers, as this will decrease your reaction efficiency and lead to unspecific products.
thanks kris, i tried on multiplex for to primers separate at one reaction using standard master mix 2X then i using 95C for 5 min (initial den), 94C for 30sec (denaturing), 55C for 30sec (annealing), 72C for 30sec (extaning), and 72C for 5min (final ext) for 35 cycles. i appled these conditions on all primers at same time. i get perfect results with sharp bands and no primers dimers.....
Great. If I were you, I would now start with a duplex, and then add more primers. This way you will see when something goes wrong.
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