Week 1 Lab: Pipetting

Contents

• Dilution Practice
• Lab

Practice

Dilution practice 1

The stock concentration of a mystery substance (MS) is 5 M. Calculate how to dilute to 100 µM (0.1 mM).

$$ C_1 V_1 = C_2 V_2 $$ $$ (5,000,000 \mu M)* V_1 = (10,000 \mu M)* (500 \mu L) $$ $$ (5,000,000 \mu M)* V_1 = 5,000,000 \mu M * \mu L $$ $$ V_1 = 1 \mu L $$

$$ V_{diluant} = V_2 - V_1 $$ $$ V_{water} = 500 \mu L - 1 \mu L $$ $$ V_{water} = 499 \mu L $$

$$ C_2 V_{2B} = C_3 V_3 $$ $$ (10,000 \mu M)* V_{2B} = (100 \mu M) * (100 \mu L) $$ $$ (10,000 \mu M)* V_{2B} = 10,000 \mu M * \mu L $$ $$ V_{2B} = 1 \mu L $$

$$ V_{diluant2} = V_3 - V_{2B} $$ $$ V_{water2} = 100 - 1 \mu L $$ $$ V_{water2} = 99 \mu L $$

For the first step, I would use 1 µL of the stock solution diluted into 499 µL of water to make 500 µL of a 10,000 µM solution. Then for the next step, I would use 1 µL of the 10,000 µM dilution, diluted into 99 µL of water to make 100 µL of a 100 µM solution.

Dilution practice 2

1. The stock concentration of a mystery substance (MS) is 5 M. If the molar mass of MS is 532 g/mol, what’s the concentration of the stock concentration in g/mL? $$ 5 M = 5 \frac{mol}{L} $$ $$ 5 \frac{mol}{L} * 532 \frac{g}{mol} = 2,660 \frac{g}{L} $$ $$ 2,660 \frac{g}{L}* \frac{1L}{1,000 mL} = 2.66 \frac{g}{mL} $$

2. You will perform a serial dilution to get 100 uM of MS. Devise a plan to dilute a 5 M MS solution to 100 uM. How many dilution steps will we need? Which tubes should we use? Which pipettes?
   We will need two empty microtubes. For the first step, we’ll use a P20 for the stock solution, and a P1000 for the water. For the second step, we’ll use a P20 for dilution 1, and a P200 for the water.

graph LR;
  A[stock solution 5M] -->|1µL stock into 499µL water| B[dilution 1: 10,000µM]
  B -->|1µL dilution1 into 99µL water| C[final dilution: 100 µM]

4. Fill out the following chart to prepare a final reaction with 60 uL reaction volume. Why did we make 100 uM MS if we actually need 40 uM MS? Why not prepare 40 uM in serial dilutions?

$$ C_{dye stock} V_{dye} = C_{dye final} V_{total} $$ $$ (6X) V_{dye} = (1X)* (60 µL) $$ $$ V_{dye} = \frac{60}{6} µL = 10 µL $$
$$ C_{MS stock} V_{MS} = C_{MS final} V_{total} $$ $$ (100 µM) V_{MS} = (40 µM)* (60 µL) $$ $$ V_{MS} = \frac{40*60 µM µL}{100 µM} = 24 µL $$
$$ V_{total} = V_{dye} + V_{MS} + V_{d H_2 O} $$ $$ 60 µL = 10 µL + 24 µL + V_{d H_2 O}$$ $$ V_{d H_2 O} = 60-10-24 µL = 26 µL $$

If we had 40 µM MS, then when we added the loading dye, it would be diluted below 40 µM. So we need to have a high enough concentration of MS, that we can add loading dye to 1X concentration and still reach a final MS concentration of 40 µM.

Lab

Part 1: Mixing Color

I made my stock color solutions by adding dye to approximately 5 ml water in three different 12 ml test tubes: 3 drops of yellow dye, 1 drop of blue dye, 2 drops of red dye, and then vortexing to mix.

Following the protocol, I obtained 6 colors. Step 4 was done with P20 and P200 in steps as described; steps 5 and 6 were done in single steps with the P1000 and P200 respectively.

I made an additional 4 colors as follows:

7. Lime: 300 ul yellow, 50 ul blue
8. Teal: 25 ul yellow, 600 ul blue
9. Coral: 300 ul red, 50 ul yellow, 25 ul blue, 300 ul water
10. Slate: 100 ul red, 300 ul blue, 300 ul water

My step 7 artwork is below and also the above cover image.

Part 2: Performing Serial Dilution

I don’t know what the Mystery Substance (MS) is supposed to be. I used some purified pUC19 plasmid, at a concentration of 197 ng/ul because that’s something I had available. It’s a double-stranded DNA, so the molecular weight would be around 660 g/mol per base pair, or a total of $660 \frac{g/mol}{bp} * 2.7 kb = 1,800 kg/mol$ approximately. Therefore, my stock concentration is $ 0.197 \frac{g}{L} * \frac{mol}{1,800,000 g} = 1.094E-7 mol/L = 0.11 uM = 110 nM$.

To get an arbitrarily chosen 1 nM stock, I did the following serial dilution:

$$ C_1 V_1 = C_2 V_2 $$ $$ (110 nM) V_1 = (10 nM)(50 ul) $$ $$ (110 nM) V_1 = 500 nM*ul $$ $$ V_1 = 4.5 ul $$

graph LR;
  A[0.11 uM stock solution ] -->|4.5 uL stock into 45.5 uL water| B[dilution: 0.01 uM]

Then I made the final solution according to the table. Again, the MS desired concentration was chosen arbitrarily.

I added 20 ul of the final solution to an agarose gel (1% w/v). I made the agarose gel by measuring out 0.5 g of agarose, and adding it to 50 ml of 1x TAE buffer, then microwaving until melted. I poured it into a gel mold with a well comb and let set fully before putting into the electrophoresis set-up to practice loading into a well.

Week 2 Lab: Gel Electrophoresis Art

Planning Notes:

• i don’t have lambda DNA, but i do have Escherichia coli BL21 genomic DNA and a small collection of various plasmids and PCR products of varying rates.
• we also have a handful of restriction enzymes but not a lot, and mostly not common ones.
• i think my strategy is going to be:
  1. sketch out a design
  2. run a restriction digest on the E. coli genomic DNA to get a bunch of different-sized fragments. doesn’t particularly matter which one i think.
  3. run the digest on a gel, and purify out the fragments of the size i want with a Qiagen or NEB kit; note: i am going to have to elute with pretty small volumes to keep them concentrated enough to show up in subsequent gels.
  4. run a new gel with the purified fragments based on the design (possibly augmenting with PCR products if desired for brightness/intensity).
  5. take photo to show off
• for whatever reason, neither uploading Genbank files and downloading accession files for the E. coli genomic assembly in Benchling is working for me. i suspect it probably has to do with the size of the files and speed (or lack thereof) of my internet. so i can’t do much in-silico planning and testing. but i think my plan will work without it. it just means i’ll have to do more testing during instead of thinking/planning prior.

Lab Prep:

1. Sketch out a design.
   I found a photo of the Portland skyline with Mt. Hood in the background from the City of Portland’s Instagram. Photo credit: @james.is.jumbled. I traced the lines of primary visual components to get a line art style drawing, and then split it into a grid of 16 columns, for the 16 wells for the largest gel comb I have available. I recreated the gridded line art, scaled to a printout of the 1kb+ gel ladder, to approximate the size DNA fragments I would need in each column.

2. Restriction digest E. coli gDNA.

   • 10 ul E. coli BL21 gDNA (125 ng/ul)
   • 5 ul rCutSmart buffer
   • 1 ul MspI (2018)
   • 1 ul SpeI-HF (2015)
   • 1 ul XbaI (2015)
   • 2 ul NdeI (2009)
   • 34 ul ultra-pure water
   • Combined the above components in a microtube (50 ul total reaction volume) and vortexed to mix. Incubated at 37C for an hour. Note that all enzymes are from NEB and are all past their expiration dates, but have been stored in a -20C freezer the whole time.

3. Gel purification of DNA fragments.
   I re-used an old gel for this first run. I combined 3ul of ladder with 2ul SYBR Green I dilution (diluted 1:50) and around 0.5ul loading dye on a scrap of parafilm, and loaded this mixture into well 5. I added 6ul of SYBR Green I dilution into the restriction digest along with 10ul of loading dye. I loaded around 33ul each into 2 lanes. I ran this electrophoresis for 40 minutes at 180mV. Lanes:

4. 1kb+ ladder (NEB)

5. Multi-enzyme digested E. coli gDNA

6. Multi-enzyme digested E. coli gDNA

7. PCR product

8. PCR product

9. PCR product

It was just smears, which I suppose isn’t too surprising, considering that I started with gDNA and all my enzymes were expired. From this gel, I cut out smears from the multi-enzyme digests at the following ranges: 1-0.1kb, 0.7-0.1kb. Using a Qiagen Qiaquick gel purification kit, I purified these semars individually. All purifications were eluted with 30 ul of elution buffer. I added these to tubes of PCR products for my gel art palette.

To another gel, I loaded the following into the wells, mixing each with 1ul of loading dye on parafilm prior to loading:

1. ladder
2. 10ul A
3. 2ul A
4. 1ul A
5. linearized plasmid
6. 15ul B
7. 2ul B
8. 1ul B
9. 5ul A

These are not super clear, but I cut out additional smears from lanes 1, 6, and 9 at the following ranges: 0.5-0.1kb, 0.2-0.1kb. Eluted these with 25ul of elution buffer. Added these to my palette above: tubes .

This left me with the following palette (all sizes and size ranges are approximate):

• A. PCR product: 6kb
• B. PCR product: 5kb
• C. PCR product: 4kb
• D. PCR product: 3kb
• E. PCR product: 1kb
• F. PCR product: 700bp
• G. PCR product: 650bp
• H. PCR product: 500bp
• J. PCR product: 200bp
• K. PCR product: 100bp
• L. smear from 100bp-1kb
• M. smear from 100bp-700bp
• N. smear from 100bp-500bp
• O. smear from 100bp-200bp

Note that J and K are low concentration, and the smears didn’t show up well on the test plate, so I’m going to use larger volumes of those than I am for the rest.

I re-drew my gridded lineart with the PCR products that I know I have.

Gel Art lab

I cast a new electrophoresis gel by dissolving 1.3g agarose in 130ml 1x TAE, and pouring into a larger gel mold. This fit a comb with 16 wells. I allowed this to set before transferring into an electrophoresis set-up filled with 1x TAE. I loaded the following combinations into the wells, mixing each on parafilm with both 2ul of SYBR GreenI (50x dil) and appropriate volumes of loading dye, prior to loading. PCR products were 2ul each, except J and K which were 4ul each. Smears (L, M, N, O) were all in the range of 4-10ul per well.

1. ladder
2. empty well
3. E, L, I
4. J, O
5. H, N
6. D, I, O
7. C, F, M, H, I
8. B, F, M
9. A, J, O
10. B, I, O
11. C, J, O
12. D, F, M
13. J, O
14. G, N, I
15. H, N, J
16. empty well

Ran gel at 200V for around a half hour.

Not all the bands are the same brightness, which I can probably attribute to the variable DNA concentration of my various PCR products. It also looks like I must’ve mixed up the 4kb and 5kb tubes. None of the smears showed up at all, which was a little disappointing. Overall though, the art turned out pretty well, I think, even if it was more trial and error than in-silico design and then execution.