User:Anthony Salvagno/Notebook/Research/2010/01/20/Preplanning for Jasper PCR
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Resuspending Oligos
I will want 100uM DNA solutions which is 100pmol/ul. Here is my information: {{#widget:Google Spreadsheet |key=tVpgQJJU0zls4LoU2qMT-NA |width=800 |height=300 }} So if you notice on the sheet, I made a mistake. I added the volume I was supposed to add to R3 to F5 so essentially I halved the concentration. Another thing to note, my nanodrop readings were way off. Somehow I got the right concentrations at the end. I don't know how it worked out, but it did. Good for me.
Protocol for Fang
Essentially I used the nanodrop (which measures absorbence of light to determine how much DNA is in solution). Using various formulas (mostly division) I obtained the numbers you see in the spreadsheet above. Since I want 100uM solution I basically divide my pmol by 100 to get the amount of ul I need to add to my DNA powder. The information for pmol and ug comes from some data sheets that Alpha DNA kindly provides.
When I add my buffer (0.1x Tris in this case) I swoosh the buffer up and down to get the DNA into suspension in the buffer. Then I just pipette 1ul out and place on the pedestal of the nanodrop. I make my measurements and record the data. Then I do the calculations you see above and voila!
Next to set up the PCR...
PCR Preplanning
BTW, I am using abbreviations for my primers. Example: pbrF2825-Cy3 is shortened to F3 for quick writing. So my primers are: F3, F5, R3, F5. {{#widget:Google Spreadsheet |key=tcRyQc0-hH9AUA1Bv0jU4_g |width=800 |height=300 }} Take note of the PCR settings table. I don't really know what my times should be but I'm going with 30s annealing and 10s extension. The rule of thumb is 1min per 1kb but I have about 100kb so that is 6s and I upped it to 10 to be safe. I feel like 30s annealing is too long, but who knows. Maybe I should up that to 1min like I usually do. I'll decide tomorrow.
SJK 11:58, 21 January 2010 (EST)
I've never done anything this short. Can you find a published protocol for an exact reaction that is this short? 10 s extension sounds too short to me. Maybe 30s? I don't think I've seen less than 1 min. maybe this page is a good starting page? Also important to consider: for short products, your primer is a bigger fraction of the final product. Thus, the amount of primer could be limiting. with 1 kb or 4kb products, we don't normally have to worry about this. For example, 100 ng / ul of final product would require 1.5 uM of primers. That's OK if you start with 10 uM I think.
