still stuck

I'm still stuck in the first step!!! There's two more weeks to ASM and I'm still struggling with the PCR. I definitely do not want to leave it hanging before going home to Penang. Will I make it in time for the wedding? I sure hope so because I don't want to leave the DNA naked at -20^oC. It’s going to deteriorate and by the time I come back, I might have to make new ones. That means all my work for one month would be gone just like that. I do need to complete the plasmid construct before I go back. Need more time!! Need to work harder!! *Red light flashing madly*

Now waiting for a PCR reaction with less amount of template and Taq polymerase. According to Kwi, I might have added too much polymerase since it also has 50% glycerol, which might affect the reaction. However, I am already using way less than 10% of the reaction volume. This is so frustrating. It’s not like I haven’t done PCR before. I’ve done it countless times and I’ve even troubleshoot it for different kinds of amplicons and templates when I was working with Sinorhizobium meliloti. Unfortunately, I have yet to work with such long amplicon and it seems like this is a lot harder to troubleshoot. Anyway, I am also running a positive control, in which I use a Verrucomicrobium genomic DNA as the template to amplify for the 16S rRNA gene. This should work since it works for everyone and I’ve used the same primers to amplify 16S rRNA gene of H. hepaticus when I was in the Young lab.

Gonna go eat my dinner for now and hopefully one hour later, I will get some products in the reaction tubes. Will have to wait till tomorrow to run the gel as the last bus is at 10pm and the gel is gonna take at least two hours.