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H . T . G . A . A .   |   WET LAB ASSIGNMENT   |   WEEK 2

BIO - DESIGN

Lab Instruction: We will be working with Addgene plasmids pPSU1 and pPSU2 deposited by the Tan lab at Penn State University. In a 2017 paper, Henrici et al demonstrated how their plasmid designs can produce cheap DNA ladder. Note, DNA ladders are important references when measuring the size and topology of DNA products after an enzymatic reaction. The enzymes we will use for our example lab are restriction enzymes, which cut DNA at sequence-specific sites. Restriction enzymes are among the natural defense systems of bacteria to protect against viral bacteriophages. They were harnessed in the 1970's as a foundational technology for mapping and recombining DNA across all forms of life. Taking part in their characterization, George published a 1978 paper that sequenced the plasmid pBR322 to help determine the recognition sequence for the restriction enzyme AvaII.

What software are you using to read the sequence files?
Benchling

What are the distances between the PstI (CTGCAG) sites in each plasmid?
500, 2000, 1000, 700, 800, 900

What are the distances between the EcoRV (GATATC) sites in each plasmid?
500, 1000, 1500, 2000

What gene is encoded into the plasmids?
ampR > which confers resistance to antibiotic ampicillin

The entire experience was very much about just being cautious and following instruction given by our lovely TAs.

It focuses on helping students grasp the basic knowledge of synthetic bio and some of the workflows and tools within the wet lab. As someone who's worked on malaria diagnostics in wet lab for a semester as a designer, I was both excited to be back in the lab, as well as anxious about finally officially learning the lab protocols, a.k.a. the correct way to do stuff.

Step 1: Harvest pPSU1 and pPSU2 DNA from overnight E. coli culture using the ZymoPure Plasmid Miniprep Kit and elute with water.

The following protocol is copied from Zymo Research

From the freezer, collect pelleted overnight liquid cultures. They are labeled "pPSU1 α" and "pPSU2 α", as the growth strain is DH5alpha. Each pellet was prepared by spinning 1.5 mL of overnight culture at 8,000 rpm (6,800 x g) for 3 minutes.

To each pellet, add 250 µl of ZymoPURE™ P1 (Red) to the bacterial cell pellet and resuspend completely by vortexing or pipetting.

Add 250 µl of ZymoPURE™ P2 (Green) and immediately mix by gently inverting the tube 6-8 times. Do not vortex! Let sit at room temperature for 2-3 minutes. Cells are completely lysed when the solution appears clear, purple, and viscous

Add 250 µl of ice cold ZymoPURE™ P3 (Yellow) and mix thoroughly by inversion. Do not vortex! Invert the tube an additional 3-4 times after the sample turns completely yellow. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form.

Incubate the neutralized lysate on ice for 5 minutes.Centrifuge the neutralized lysate for 5 minutes at 13,000 rpm (~17,900 x g).

Transfer 600 µl of supernatant from step 6 into a clean 1.5 ml microcentrifuge tube. Be careful not to disturb the yellow pellet and avoid transferring any cellular debris to the new tube.

Add 275 µl of ZymoPURE™ Binding Buffer to the cleared lysate from step 7 and mix thoroughly by inverting the capped tube 8 times.

Place a Zymo-Spin™ II-P Column in a Collection Tube and transfer the entire mixture from step 8 into the Zymo-Spin™ II-P Column.Incubate the Zymo-Spin™ II-P/Collection Tube assembly at room temperature for 2 minutes and then centrifuge at 6,000 rpm (~5,900 x g) for 1 min. Discard the flow through.

Add 800 µl of ZymoPURE™ Wash 1 to the Zymo-Spin™ II-P Column and centrifuge at 6,000 rpm (~5,900 x g) for 1 min. Discard the flow through.

Add 800 µl of ZymoPURE™ Wash 2 to the Zymo-Spin™ II-P Column and centrifuge at 6,000 rpm (~5,900 x g) for 1 min. Discard the flow through.

Add 200 µl of ZymoPURE™ Wash 2 to the Zymo-Spin™ II-P Column and centrifuge at 6,000 rpm (~5,900 x g) for 1 min. Discard the flow through.

Centrifuge the Zymo-Spin™ II-P Column at 13,000 rpm (~17,900 x g) for 1 minute in order to remove any residual wash buffer.

Transfer the Zymo-Spin™ II-P Column into a clean 1.5 ml tube and add 25 µl of DNase/RNase-Free water directly to the column matrix. Incubate at room temperature for 2 minutes, and then centrifuge at 13,000 rpm (~17,900 x g)for 1 minute in a microcentrifuge.

The plasmids were already incubated with e. coli and we had to each individually carry out the process of DNA isolation. I was most worried about pipetting the incorrect amount or contamination, or messing up the concentration ratio or the mixing process.

3. Measure the concentration of your pPSU1 and pPSU2 DNA preps using the Nanodrop spectrophotometer with the following steps.

Clean stage with Kimwipe soaked with DI water.

Wipe stage with dry Kimwipe.Add 2 uL DI water on stage and make blank measurement.

Wipe stage with dry Kimwipe.For each DNA sample, add 2 uL and measure and record the concentration (ng/uL).

Wipe stage dry with Kimwipe between measurements.

This was rather straight and forward. The measurement software showed expected graphic results, yet the amount of DNA were potentially problematic. I had a concentration of 37 and a concentration of 59, when the procedure should leave me way over a couple of hundred if not over one thousand.

The cause of this result was likely due to extract the cleared lysate from the centrifuged tubes. The RPM of the centrifuge seemed a little bit off, and I was left with floating cell debris in the concentration, which made the extracting process a harder.

The rest of the lab focuses on measuring and creating the different solutions used for each slot in the gel box with the diluted DNA cut / uncut samples. Working with the thermal controller was a little difficult because as a programmable machine with no explanation of procedure, it's hard to know if we've adjusted to the temperature or time right. Fortunately, we had help from out bio-engineering teammate.

4. In separate digestion reactions with either the NEB enzymes PstI-HF or EcoRV-HF, cut both pPSU1 and pPSU2 DNA.

If plasmid yields are greater than 500 ng/uL in concentration, prepare a dilution of your plasmid with water to a concentration of 100 ng/uL and total volume greater than 10 uL. If a plasmid yield is less than 2 ug in total yield (concentration x volume), either repeat the prep or use as much as you have available for the two digestion reactions and uncut controls.

Incubate reactions at 37C for 15 minutes in thermocycler.

There was a lot of math.

4. Run the reactions alongside 100bp and 1kbp DNA ladder and uncut plasmid controls on a 1% agarose gel stained with SYBR-Safe at 120V for 1 hour. Scale down steps (i) and (ii) below by 1/3 if you are using the smaller gel box.

Weigh 1.5 g agarose and add into 150 mL 1xTAE. Microwave until agarose completely dissolves.

Add 15 uL of 10000xSYBR-Safe DNA stain. Use heat-resistant glove to gently swirl to mix (careful of burn hazard).

Pour into rotated gel box tray (check beforehand that seal is watertight and do not fill past the bottom of the comb slots) and insert comb.

Wait 45-60 minutes for gel to set.

Remove comb and rotate tray so lanes are closest to cathode (black cable). Fill gel box with 1xTAE until gel is submerged.Load the following into separate lanes.

Insert gel box lid, connect cables to power supply, and run at 120V for 1 hour, and then image the gels with a transilluminator and analyze the banding patterns.

The left shows two sets of experimentation results from our team (6 each). The results were fairly accurate compare to the standard, through there were some smudging, that may be caused by rotating the gel before running the box (as our original casting orientation was sideways) or accidental puncturing during the loading process.

I'm still somewhat confused about parts of the process and the reasons / reaction behind the procedures, but I guess it helped a lot in terms of getting used to the wet lab standard procedures, equipments and general expectation of bio-engineering experiments.

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