Mrenrisco

Hi guys, so I started my PCR last week, I'm analysing capsicum species using RAPD markers, but I'm a little confuse of how should I procede with my PCR. First of all, my professor said that I couldn't make the mix all at once and then distribute to micro tubes with my DNA samples, she said that I should prepare the mix separately. Well that's quite annoying, because I have a lot of samples and I must amplify each primer 3 times. Is it really necessary prepare the mix for each sample? Second, for my project my professor selected 20 primers to analyze the polimorphism and etc. will each primer have its own temperature of annealing, desnaturation etc. or should I configure my termocycler once and use the same program for all primers? and last but not least, this was my first electrophoresys of a PCR: this was just a test, I used 11 samples: some of them amplified, some of them not... but I'm not actually worried about that, I want to know why those wich amplified didn't show clear bands, and why my blank (the sample right before the ladder) stained. Oh, I used the primer OPA 020 for this first test

oh thanks, I'll try to be more careful when pulling out the comb. But this marker is old, my professor asked me to test it, that's why I let it run for a short time.

thanks for the answers "pocket for the marker seemed to be damaged" could you explain it more precisely?

Hi everyone, I'm an undergraduated student and I have been extracting DNA from seeds successfully for a couple of weeks. But when I try to visualize the quality of the DNA extracted using agarose gel with BrEt, my gel gets orange. The DNA was extracted according Doyle & Doyle protocol. I'm adding 3ul of BrEt for each 50ml of agarose (I'm adding BrEt in the gel). This souldn't happen. Can anyone help me? I'd like to solve this problem before my professor request me to show my results As a loading buffer I'm using a solution with bromophenol blue and sucrose