I have tried standard barcoding primers LCO1492 and HCO2198 but it didn't work. Has anyone worked on polychaetes barcoding?
Hey Reena, Hiroshi is correct the primer is LCO1490 and not 1492.....
I did my honours project on polychaetes last year and i used LCO1490 and HCO2198 and they worked very well for the entire class. Each family has a different annealing temperature. For Nereids in particular, they annealed at 45 degrees Celcius. Also i used the following PCR protocol: 40 cycles of (95 °C for 3 minutes, 94 °C for 30 seconds, 45 °C for 30 seconds, 72 °C for 1 minute) followed by an extension of 72 °C for 10 minutes and 12 °C for infinity. If you happen to get double bands, you can do a reamplification technique by poking the desired band region (~600bp) several times and leaving it in a pcr tube containing master mix and primers for about 5-10 minutes. Then you have to run a pcr again and you will get a very bright single band beacuse you are only reamplifying that one particular fragment of ~600bp.
If you are unsure, you can maybe do a gradient PCR from 40-50 and see where that takes you. But i am very certain about my protocol because i got good results.
Anyways if you do happen to try my method, please let me know how it goes.
Goodluck and all the best!
Jyothi
I have not tried on Polychaetes but JB3 and JB5 primers works well on Nematodes barcoding. You may check the specificity of those primers in Polychaetes. I dont remember if those primers are specific to Nematodes as I have done long back. You may give a look..
What polychaete group (e.g., family, genus, or species) do you deal with? That information would help us answer the best solvent.
By the way, one of my colleague who study the taxonomy of capiteliid polychaete also has tried to amplify the COI gene sequence using LCO1492 and HCO2198 primer. Then, it succeeded. So, I guess that these primers can be applied for almost all (but maybe not all) polychaetes. Do you have any other reasons why you cannot succeed PCR? (e.g., DNA has already been broken by hydrolytic degradation)
hi Hiroshi Yamasaki
i am dealing with nereidae polychaetes n when i am trying to amplify co1 with LCO1492 and HCO2198 primer i am getting double band i have tried by increasing the annealing temp. but then also i didn't get a proper band. the quality of dna was also good.
I found a several papers which include some nereidae polychaete, and they used HCO2198 and LCO1490, not LCO1492. Is LCO1492 your mistype?
Anyway, it is mysterious why you can not amplify with LCO1490 and HCO2198.
How about try again with lower annealing temperature, or try to design a new primer for nereidae polychaete? You may be able to design it based on other sequences data registered in Genbank.
Hey Reena, Hiroshi is correct the primer is LCO1490 and not 1492.....
I did my honours project on polychaetes last year and i used LCO1490 and HCO2198 and they worked very well for the entire class. Each family has a different annealing temperature. For Nereids in particular, they annealed at 45 degrees Celcius. Also i used the following PCR protocol: 40 cycles of (95 °C for 3 minutes, 94 °C for 30 seconds, 45 °C for 30 seconds, 72 °C for 1 minute) followed by an extension of 72 °C for 10 minutes and 12 °C for infinity. If you happen to get double bands, you can do a reamplification technique by poking the desired band region (~600bp) several times and leaving it in a pcr tube containing master mix and primers for about 5-10 minutes. Then you have to run a pcr again and you will get a very bright single band beacuse you are only reamplifying that one particular fragment of ~600bp.
If you are unsure, you can maybe do a gradient PCR from 40-50 and see where that takes you. But i am very certain about my protocol because i got good results.
Anyways if you do happen to try my method, please let me know how it goes.
Goodluck and all the best!
Jyothi
also you can use 1 microlitre of BSA (bovine serum album) in your pcr mastermix. that works wonders!!!!
Can I buy LCO1490 and HCO2198 it directly from a dealer or I have to synthesize it?
Hey Mauro, yes you can buy those primers directly from the company....
Jeffrey, there has been a publication on new primers available for barcoding polychaetes ... i will attach the paper and you can read through it.
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