Hello dear colleges!
Well, I have this issue that are driving me crazy. Im trying to amplify COI Folmer Tailed HCO/LCO primers for Barcoding, but I have this faint bands (Attached file). I already try changing the Thermal Cycler (in fact I already used three different thermal cyclers), PCR conditions, PCR program, Quantity of DNA, Quantity of primers, MgCl, temp grad, and more... The DNA is working because I already amplified 12S and 16S
Other thing is that I don't know why my ladder is faint too, I already bought a new one and is the same, I used another power supply and different % of agarose and nothing change, we are using Et-Br, because we're out of gelred stock.
Any suggestion?? I'm little stressed about this :(
Thank you everybody!
I do not think that the problem is faint bands. Ifyou think that they are faint then one possibility is that the filter on your transilluminator system is wrong or you are taking too long to take the picture and the signal is bleaching . The main problem is that your samples are running badly and smearing instead of running in a tight band. You may have too much salt in your samples or loading dye but I would try letting the gel set for longer, adding slightly less loading dye and running at a lower voltage for a longer time ( ideally in a cold room to keep the gel cool but this is not essential)
Thank you very much Dr. Rutland. I'll try adding less loading buffer (I use 1ul per sample). I tried with 100volts/400mA for an hour and is the same result, just the ladders spared more than usual and yes, I have the smearing too. I'll check the transilluminator conditions to0, to get better results in my gels.
I think there might be a problem with running buffer. Use freshly prepared 1X TAE ( use Tris Base not Tris buffer while preparing buffer).
How long is your gel please?
I would run at less than 5v/cm so suggest you try 3.5 v/cm of gel which for a 10cm gel would only be 35v ( 70v for a 20cm gel) and make sure that the oncentration of TBE ( or TAE) in the gel is the same as that of the running buffer.
best wishes
paul
You need to run on 70 volt and and Use 0.5X TBE buffer for running gel and Making 1% agarose 0.5X TBE. It's take time half an hour to one hour.
best wishes
Ashish
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