User:Anthony Salvagno/Notebook/Research/2010/01/28/Jasper PCR 5.0 Results
Imagine my surprise when I saw that under the transilluminator! Anyways I'm not going to post another shitty gel image (actually the gel came out rather nice, but the results not so much). Instead I am going to focus on how to make this reaction better and then do that.
I'm low on Taq and I don't trust my buffer and MgCl2 and dNTP's anymore (despite the 1 working well reaction), so I think I want to buy new stuff.
- New Taq
- New PCR buffer
- New MgCl2
- New dNTPs
- New Thermal Cycler
I will also read some PCR protocols to try and figure this thing out. I'll post ideas at some point.
PCR Potential Problems/Fixes
Ramalldf 06:07, 29 January 2010 (EST)
- That stinks. If it makes you feel any better, my PCR amplification hasn't been working either. I ordered some new primers last night and will try those tomorrow. And we have a very crappy thermocycler too that has the heater for the lid as a separate instrument. Apparently it's coal-powered.
- Is there a reason why you can't get this ruler synthesized? You guys use AlphaDNA right? I don't know what their prices are like, but I just checked the prices at IDTDNA and for 25
nmnmol of custom DNA, it is about 35 cents/bp. For you it would be about $35, which is probably cheaper than the amount of money that has been used up in reagents and your time thus far. I think that those are the ssDNA prices, but if you already have primers, maybe that will make the process a little simpler?
- Steve Koch 12:37, 29 January 2010 (EST): Thanks as always for the advice, Diego! I had a hilarious dream that I need to tell you about, but it's not PC enough for OWW. Anyway, as for synthesizing, they had actually tried this method before, and it was not working because of yield issues. Maybe IDT can do long oligos better, but I sort of doubt it? In any case, I'm sure Ant will get it to work. My gut is that it's our barbie PCR, so hopefully it will work better in Osley lab.
- Ramalldf 03:14, 30 January 2010 (EST):That's funny, which reminds me about that dream I had a few years ago about the robot we made out of the optical trap in order to catch food since the lab was stranded on a deserted island :D
- Lastly, does it have to be a specific 100 bp sequence or even exactly 100 bp? I'm sure that someone has some plasmid out there that you could use that will end up giving you a hundred bp fragment? If this isn't a crazy proposition, I'd ask Kelly/Osley what vectors they had and then look at the multiple cloning site of these (MCP, has unique sites here for cloning) and find which one will suit you best. Then transform this into bacteria, miniprep to get lots of DNA and finally digest it and gel purify. Good luck.
- Steve Koch 10:19, 30 January 2010 (EST): Given the top-secret aspect of this project, you may have missed that the primers are labeled with Cy3 and Cy5 -- the plasmid method will work, with ligation of hairpins (like you did with biotin / dig tagging a long time ago). But we chose PCR (like you also did with dig / bio) because it is "easier." Diego made me remember a good point, though. We figured it'd be easy enough that you just designed a new reaction on pBR322. It's probably too late to copy an exact reaction (would need new primers). But can you find a published reaction that someone out there is using for 100 bp PCR reaction? Maybe you can see something about their conditions. But I feel there's a good shot that PCR in Osley lab on Monday will work.