Estimated g023 P159/170 PCR product concentration based on gel from yesterday. Probably around 7ng/ul. Annealed 2x20ul of each product, didn't add anything or purify, according to the SURVEYOR protocol. Cut with 2ul SURVEYOR nuclease and 1ul of enhancer. Used 3ul of elution solution for -nuc samples. Incubated at 42 for one minute, then it went back to room temp because I set the new PCR machine incorrectly. Noticed after about 10-15 minutes, incubated for another hour.
Added 1ul of cut stuff to 2ul stop solution and 17ul elution solution. Put at -20
Didn't add stop solution to the rest, just directly analyzed on 1.5% agarose gel. Did some reading on agarose gels as well:
- voltage should be 4-10V/cm according to the distance between the electrodes. Our small gel box distance is 16cm so I will run at 5cm/V, or 80V.
- gels that are too thick will blur the bands. The thinnest gel comb we have is 0.1cmx0.4cm. I will have about 23ul of sample-1ul for bioanalyzer+4.4ul 6x loading dye or 0.0264cm^3. To determine the height of the gel I should use to fit all my sample in, I divide by the area of the comb.
- 0.0264/0.4/0.1= 0.66cm
- to determine the volume of gel that corresponds to that gel height, i use the length and width of the gel cast tray: 11cmx9cm
- Cleaned the casting tray and the combs.
- I just poured a 60mL gel because I did the calculations wrong and then added 5mL of TAE while it was cooling. Used 6.5ul SYBR Safe
- A 1.5% gel should be good for resolving the smaller bands
- Will run for 45 minutes