Lab 4 Procedure
Determining the DNA Concentration:
- Take out the purified PCR products and EB from the freezer box.
- Once thawed, you will determine the concentration of the PCR products using a NanoDrop spectrophotometer.
- Here is a video showing how to measure DNA concentration on a NanoDrop spectrophotometer:
- Add 2 μL of EB on the platform of the NanoDrop.
Make sure the EB (and DNA samples) is completely thawed before use. Otherwise, you will obtain incorrect concentration.
- Click on “Blank” on the program on the computer. This can take up to 10 seconds.
- Once the blank has been completed, add 2 μL of your purified PCR product onto the platform and click “Measure”.
- Write down the DNA concentration and the 260/280 and 260/230 ratios.
- Repeat with the rest of your samples.
Calculate and Assemble the YAC Reaction
- For the upcoming YAC experiment, you will need 0.05 pmol of DNA for each of the PCR products that you obtained.
You must use the same pmol for all PCR products! If you have samples that are less than 0.05 pmol, please discuss with the teaching team.
- Determine how much each PCR product is to use.
- Working in a group of 4, calculate the pmol of each PCR product.
- To calculate the pmol, first, convert the concentration from ng/ μL to μg/ μL.
- For example, if you have a 1000 bp DNA with 50 ng/ μL concentration, then first, convert the concentration from 50 ng/ μL to μg/ μL by dividing the concentration by 1000, so 50/1000 = 0.05 μg/ μL
- Then, plug in the number using the formula below
- N = number of DNA in bp (you can find the expected fragment size in bp in the Appendix)
- So, to continue the example, 0.05 * 1/660 * 10^6 * 1/1000 = 0.076 pmol/ μL
- So, to use 0.05 pmol of the DNA, then one should use 0.05/0.076 = 0.6 μL of the DNA sample.
- Since this amount is too small to pipette, a dilution should be made. In this case, one can make a five times dilution with 1 μL of the DNA and 4 μL of EB for a total volume of 5 μL.
- One can then add 3 μL (equals to 0.6μL in the original sample) to make the DNA mixture.
- Once you calculated the amount needed, take out 1 microfuge tube and assemble the following DNA mixture for a final volume of 34 μL:
- ___ μL F1
- ___ μL F2
- ___ μL F3
- ___ μL F4
- ___ μL F5
- ___ μL P1
- ___ μL P2
- ___ μL P3
- ___ μL H20 (add up to 34 μL)
- Label the tube and store the tube in your freezer box for the next lab.