ocean_sky83

When I run my gel, I noticed a very faint band, I almost missed it. I do not know yet  whether it could be because of "carry-over" from the well beside it. If it is not due to accidental carry over, I want to do some optimising to my pcr, my question is,

a) How do u tell if your bacteria dna has high GC content? How much of betaine do u usually add to your reaction tube?
If I do use betaine, is it necessary to lower or increase the annealing temperature?

I hope I am thinking too much about the faint band. It is so faint that I stared very long at the image before realizing it Do you usually follow up on an extremely faint band or just rule it out as negative, assuming you do not know whether in first place it is supposed to be present. When I observed the other positive samples I have, they are all very bright clear band. There was one faint like a "pencil line band". Could this be due to high GC content? My product size is about 700+bp I run it on a 1.5% gel.

Usually, would you repeat on such cases?

Any advices very much appreciated!!
Have a great day!

Thanks!!
Mich

Hi

I am trying to do a intergon pcr... The pcr machine I am using has this "asterisk" which indicates for the "ramp" function. I am using 68 degrees celcius for my elongation temperature.. and 4 min elongation time.. does the ramp also increases temperature after every cycle? I only know the elongation time is increased every cycle. is this optimised temperature? should i change it to 72? somehow someone has been using 68 degrees and thus i am hesistant to change the temperature as i do not know if it may be optimised. what should i do?

I am trying to do sequencing on the entire gene expecting about 4kb, thus i was advised to use 4 min elongation time.. i want to know when i get my pcr product on a gel, would it shows up around 4kb size? or roughly 1000 to 1500 bp?

Sorry if i sound confusing.
I must admit I am a really lousy in this! Sorry.. =(

Thanks