Hello,
we are currently genotyping chironomids using the classic Folmer LCO1490 and HCO2198 primers. the LCO primer always performs very good, but the HCO primers do mostly bad. We are cycle sequencing with an annealing temperature of 50°C for both primers. Did anyone have similar and solve the problem? Thank you in advance for your help!
Cheers,
Helge
can we see a ,abi sequencing file of the poor sequencing please. What method of primer removal are you using please?
Is the HCO sequence GC rich?
Why are you using 50c for sequencing...because the kit says so or is this the annealing temperature of the primers for the pcr ( I assume you use the same primers for pcr and sequencing)
(1) Do you get clean bands from your PCR? If so, consider lowering your annealing temperature. I have used as low as 45oC for mtDNA sequencing.
(2) Consider doubling your primer concentration for sequencing. A combination of (1) and (2) usually solves stubborn mtDNA sequencing issues for me.
(3) Does your LCO sequence read stretch the entire length of sequence (~700bp)? If so consider aligning some of your LCO sequences against closely related species from genebank and selecting a more conserved region to design your own species-specific primers.
Thanks for your answers!
@Paul:
(1) We are using Sephadex columns.
(2) Yes, the primers are the same. The Tm of the HCO primer is about 61°C. I was using 50°C for sequencing because I was taught to use 50°C if the Tm exceeds this temperature (probably due to the manual).
@Joel:
(1) Yes, the bands are clean. I was rather thinking about increasing Ta for the reverse primer. But I will check!
(2) I will try this tomorrow, thanks!
(3) Yes, it reads the entire length. Unfortunately we are limited in time and designing own primers is not possible at the moment.
Thanks again and Cheers,
Helge
try annealing at 60c.I have used annealing up to 64c because the primers worked and to remove secondary structure problems. If the sequencing works but stops early do the sequencing in presence of 1M betaine to remove secondary structure problems if high GC. The kits are too conservative any temperature up to 60 will work fine but like pcr the hotter the better.
Thanks Paul! I will try higher Ta today. Does Betaine affect Tm similar to DMSO? Cheers; Helge
Use the same annealing temp with betaine as without. It acts differently from dmso which effectively increases the tm of the primer binding. Just to be sure when you said you use sephadex to remove primer you did mean from the pcr mix and not from the post sequencing reaction didn't you?
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