EmpireSpit kept a very thorough lab notebook, as we thought it crucial to precisely document all of our progress each day in the lab. Initially, we made many small errors resulting in confusing results that would’ve had no identifiable cause if not for the crisp track record we were able to look back on. Additionally, it’s much, much easier to replicate experimental steps with complete step-by-step instruction. Altogether, to stay organized, keep our timeline firm, and save time in our future recreations of experiments, it was more efficient to keep a detailed history of everything accomplished in the lab.


During June, we focused on meeting requirements for getting team members into the lab and also attended an orientation.

June 26, 2023 (Biology Team Orientation)

Attendees: Christina, Seungmo, Steven
Time Present: 9:30-11:30 AM, 1-4 PM

Learned lab safety procedures, took a tour of the lab


In July, we prioritized the early stages of our project. Notably, we started developing the restriction digest for our plasmid construct.

July 9, 2023 (Lab Setup)

Attendees: Christina, Steven
Time Present: 10 AM-12 PM

Washed dishes, organized lab space, discussed lab operations for presentation on 7/10 meeting

July 10, 2023 (Biology Team Orientation)

Attendees: Daniel, Heewon, Damini, Grace, Justin, Steven
Time Present: 9:30-11:30 AM, 1-4 PM

Learned lab safety procedures, took a tour of the lab

July 13, 2023

Attendees: Daniel, Heewon, Justin
Time Present: 12-4 PM

    • LB Broth
      • Homogeneous solid powder that allows for the growth of bacterial cells.
      • Contains yeast extract, sodium chloride (NaCl = Na+ and Cl- minerals / ions), and tryptone in the right ratios for the optimal growth of the bacteria. Yeast extract, sodium chloride, tryptone in 2:2:1 is the most optimal ratio for growth.
    • Dissolve LB broth powder into MQH2O (MilliQH2O)
      • Heterogeneous solutionwith some solids in the liquid → invert / inversion, x9 → autoclave, liquid 60 cycle (to boil which further dissolves and homogenizes solution as well as sterilizes) forming LB broth without antibiotics.
    • Inversion / Inverting – to make sure none of the LB broth is stuck at the bottom or the sides by shaking the solution.
    • Autoclave – machine used to carry out industrial/scientific processes related to elevated temperature/pressure; solution must reach temperature to clock (starting the process) let chamber preheat: 30 min. Broth cook time: 60 minutes.
      • Two probes in autoclave.
        • First, in the back is to measures the temp of air within the chamber
        • Second, in the front is a silver elongated piece connected to the wire which is a probe to measure temp of solution
          • These probes are used to manage temp in autoclave.
          • Ratio to purified water = 25g /1 L milliq water (purifies water via electric current)
    • Microbiology Growth Curve in Four Steps:
      • LAG: microbes need to recover because they're frozen at -80C (absorbing nutrients). The microbes that survive are based on luck.
        Nutrients (N) >> microbes (m) OR m << N (nutrients far greater than microbe population)
      • LOG: microbes recovered, actively dividing.
        N > m OR m < N (more nutrients to consume than microbes present)
      • PLATEAU: microbes in homeostasis where nutrients consumed are equal to waste excreted AND the population is growing at a similar / same rate as the population dying.
        N = m OR m = N (rate of nutrient use = rate of waste)
      • DEATH: microbe population in decline.
        N < m OR m > N (Less nutrients to consume than microbes present)
    • Lab coats - Take from inside the closet near Allison’s office.
      Return on Friday night so they can be washed on Monday morning.
    • Scales & Weighing - Ordered from most sensitive/most accurate to least sensitive / least accurate.
      • Less accurate reading scale can measure more (25g vs 25mg) but much less accurately than the more accurate reading scale (can measure less but is much more accurate).
      • Use a weight boat when measuring powder. Dispose of the weight boat in the large sharps bin.
    • MQH2O – Electric current that must run through water for proper purification: 18.2 megaOhms (hit the button to turn water flow on and off).
      • Let the water run until 5 droplets symbol comes up, then watch the beaker level as the water fills.
      • MQH2O has all impurities removed (ONLY H2O molecules), molecules (DNA, RNA etc) and minerals (ions) are removed.
      • Difference from distilled water - water with only ions removed.
    Day 1 Experiment Summary:
      • We used a scale and a weighboat to measure 25g of LB Broth powder.
      • We used the MilliQ system to measure one L of water in a graduated cylinder.
      • We added 900 mL (.9L) of water to our 25g of LB powder.
      • We utilized the inversion technique to mix our components 9 times (an arbitrary amount).
      • We added an additional 100 mL (.1L) of water to raise the volume of water to 1L.
      • We lightly sealed the media bottle, placed a slip of autoclave tape on the cap, placed both the solution and a liquid probe in an autoclave pan, and placed the temp probe into the autoclave for 1 hour to cook / sterilize (already had been heating for 30 minutes).
    Work Summary:
      • Reviewed one glove procedure.
      • To prevent external particles from entering the erlenmeyer flasks, we placed aluminum foil over the top (they had already been cleaned). We did not put foil on the ones that were not cleaned. Need to clean glassware at the end of the week.
      • We organized the flasks in size order on the shelves.
      • Steven presented a summary in a meeting room.

Lab 1

July 14, 2023

Attendees: Daniel Yi, Heewon Choi, Justin Hong, Damini Rosenblatt, Grace Lee
Student Time Present: 12P to 4PM

    Steps for Constructing Artifical Alpha Amylase
      Molecular Cloning:
      • PCR Amplification of Alpha Amylase Insert (Gene of Interest):
        • Use PCR to generate more copies of the alpha amylase insert (GOI).
      • Gel Extraction of Alpha Amylase Insert:
        • Extract the alpha amylase insert from the PCR product.
      • Linearization of Plasmid Backbone:
        • Utilize restriction enzymes (XbaI, XhoI) to break the circular plasmid backbone at the multiple cloning site (MCS).
        • Perform gel extraction to obtain the linearized (broken) backbone.
      • Assembly of Plasmid:
        • Combine the alpha amylase gene (GOI) obtained in step 2 with the linearized plasmid backbone from step 3.
        • Allow the GOI and backbone to anneal overnight.
      • Gel Extraction of Reconstructed Plasmid:
        • Extract the newly assembled circular plasmid.

      Transformation: Insertion of Plasmid into Chemically Competent Bacteria (DH5 alpha cells):
      • Plasmid Introduction and Replication:
        • Introduce the plasmid into chemically competent DH5 alpha E. coli.
        • The bacteria replicate the plasmid, producing more plasmid DNA.
      • Miniprep to Extract Plasmid DNA:
        • Perform a MiniPrep of DH5 alpha E. coli to extract and concentrate the plasmid DNA.
        • Repeat this process as needed until approximately 100 nanograms of plasmid DNA is obtained.
      Bacterial Growth:
      • Lag Phase:
        • Take bacteria from -80°C storage.
        • Introduce the plasmid.
        • Create a 1 mL day culture with shaking in LB media without Kanamycin resistance.
      • Log Phsae:
        • Take the day culture and add 4 mL to it.
        • Allow overnight growth with shaking
        • Subsequently, utilize the MiniPrep kit to extract and concentrate plasmid DNA.
        • Make glycerol stocks for future use.

      Purpose of Bacterial Cultures
      • Made LB Broth → to grow bacteria which are currently frozen: stored -80 degrees celsius → “inactive”/”preserved” → fragile → engineered to be chemically competent - cells have been treated to have holes in bacterial cell wall:
      • Purpose of Holes in Bacteria Cell Wall
        • The bacterial cell wall surrounds the bacteria
        • Cells have been treated to have holes in bacterial cell wall
        • We have the cells chemically competent, which make holes that allow plasmid DNA to go from extracellular (outside space) to >intracellular (inside space)
      Function of both strains of E. Coli
      • DH5alpha (DH5a for molecular cloning: backbone + endonuclease + insert = plasmid)→ makes more plasmid DNA (human alpha amylase as DNA)
      • BL21 for transformation for plasmid, which came from DH5a mini prep → make plasmid DNA to alpha amylase PROTEIN
      Summary of each step in the alpha-amylase synthesis process
      • LB broth was made to grow bacteria(DH5alpha / DH5a, BL21)
        • Stored at 80 C (frozen)
        • Becomes inactive/preserved
        • Engineered to be chemically competent
        • Holes allow plasmid DNA from go from extracellular(outside) space to intracellular(inside) space
        • If plasmid (DNA) was to go into the bacterial cell wall, it cannot happen without being chemically competent
        • After it is made to be chemically competent, the plasmid can go into the intracellular space
      • We use two different lines of e coli (DH5a and BL21)
        • DH5a: to make more plasmid DNA(human alpha amylase as DNA)
        • BL21: make plasmid DNA to alpha amylase PROTEIN
      Components of the artifical alpha-amylase
      • Molecular Cloning: backbone + endonuclease + insert = plasmid DNA
        • Use PCR to make more aA gene insert
        • Gel Extraction of aA gene(1/3)
        • Use restriction enzymes(Xba1, Xho1) to break backbone at multiple cloning site(mcs): backbone is circular DNA that works in bacteria
        • Gel Extraction of "broken" BACKBONE
        • Assembly of aA gene/GOI with "broken" BACKBONE to make the plasmid
        • Gel Extration of plasmid
      • Transformation of DH5a with plasmid: makes more plasmid DNA
        • Transformation: insertion of extra chromosomal DNA(the plasmid) into our chemically competent bacteria
        • Take plasmid + kit(magic) = plasmid in E.coli(this produces more plasmid DNA within bacteria)
        • Miniprep(kit) of DH5a to extract and concentrate plasmid DNA
      • Bacterial Growth Review
        • Lag Phase: take bacteria out of the -80 Celsius, then introduce the plasmid, then make 1mL, day culture w/shaking LBmedia w/o KanR. So in terms of previous steps, we add the plasmid to the DH5 alpha E. coli which produces more plasmid and then eventually we can use the log phase to add the plasmid to the BL21 E. coli.
        • Log Phase: take the day culture + 4mL, overnight w/shaking. Afterward, we will kill all the E. coli using the miniprep kit.

July 15, 2023 (Full Team Meeting)

Attendees: In-Person Full Team Meeting
Time Present: N/A

Citations: 6 Points to Prove

  • Blood draws expensive, invasive, unpopular
  • Test kits, expensive, time-intensive/aptomer superiority
  • Cortisol, Alpha-Amylase, and Specific mRNA are detectable in stress
  • Inaccuracies with in-person diagnosis (therapist)
  • Relationship between stress, anxiety, depression

July 20, 2023

Attendees: Christina Sher, Grace Lee
Student Time Present: 1130A to 4PM
  • Using LB Agar
  • Pour 1000mL of water into 40g of LB agar
  • Put it autoclave for LIQ60 cycle with liquid probe
  • Liquid probe ensures media sterilizes and boils, which turns the agar powder into a solution
  • Washed dishes using the Steris machine
  • Take out the solution in autoclave
  • Put the kanamycin (KAN) into the agar solution when cooled to ~160°C / cool enough to hold and pour.
  • Pour into plates - preparing petri dishes

  • How much kanamycin did we add into how much LB agar?
  • What is the ratio?
  • Why do we wait to add the KAN?
  • Where did the KAN come from?
  • Where did we pour the plates?
  • How did we pour the plates?
  • Where did they stay that night?
  • Where did they move to the next morning?
  • What labels did we add to the dishes? To the bag?
  • Where will the future bacterial work be done?
Link to Document

July 21, 2023

Attendees: Damini
Time Present: N/A

  • Resuspend GOI in 20 uL H2O
  • Determine the concentration using nanodrop → 1uL h2O to blank, 1uL DNAto read = 255 ng/uL = 5100 ng
  • Make 10 uM primers for PCR for 100 uM stocks
    1 part primer: 9 parts H2O + make 10x dilution
    10 uL primer: 90 uL H2O + 10 uM dilution
  • NEB says to template DNA < 1000, is 255 ng → going to dilute to 100 ng/uL
  • NEB 25 uL reaction

July 24, 2023

Attendees: Daniel Yi, Grace Lee, Heewon Choi
Streaking for Single Colonies:
  • Glycerol stock was at 4 degrees Celsius.
  • Stabbed in the bacteria room with pipette tip.
  • Streaked the bacteria onto LB (nutrients to grow bacteria) Agar (growth matrix/foundation) with Kan (kanamycin, antibiotic, selection factor; screens out any bacteria that DOES NOT contain kanR gene: a protein that will enzymatically degrade the Kan: carbohydrate antibiotic)
  • Protect our bacteria of interest that contain the plasmid.
  • Bacteria vs. Die because no plasmid that has the KanR gene.
  • The glycerol stock (contains the plasmid PET-28a(+)) stored in the freezer at 4 degrees Celsius is metabolically inactive/inert (does that multiply).
  • We took pipette tips and left for the freezing room.
  • We went to the freezing room to get the LB Agar with Kan in the Petri dishes that were prepared last week.
  • We took it to the bacteria room and stabbed the pipette tip into the glycerol stock for the single colony growth technique Streaking for single colonies.
  • We streaked the glycerol stock onto the Petri dish that has LB Agar with Kanamycin. This allows the bacteria in the glycerol stock to become metabolically active/inert (allows it to multiply).
DNA and RNA:
  • DNA makes a copy of its instructions into mRNA (leaves the nucleus in human and plant cells and goes to the endoplasmic reticulum). In bacteria, DNA is transcribed in the cytosol.
  • The smallest living entity is the cell in biology, in bacterial, animal, and plant cells.
  • 3 RNA nucleotides = 1 codon = 1 amino acid.
  • 4 exceptions to this rule.
Additional Information:
  • Streaking for Single Colonies:
    • Glycerol stock was at 4C.
    • Stabbed in bacteria room with pipette tip.
    • Streaked the bacteria onto LB agar with kanamycin.
    • Put it in 37C incubator (not liquid stock so not put in the shaker).
  • LB: nutrients for growth.
  • Agar: growth matrix/foundation.
  • Kanamycin: it's an antibiotic → selection factor → screens out any bacteria that does not contain KanR gene.
  • KanR is a protein that will enzymatically degrade Kan (carbohydrate) antibiotic. This is to protect our bacteria of interest that contain the plasmid.
  • The point of streaking is to wake up the glycerol stock that is inert. We have to wake them up.
  • We are streaking to get single colonies of bacteria.
  • Overnight Culture:
    • Take out the colonies and put into LB broth to make the bacteria multiply more.
    • Use Miniprep to extract plasmid.
  • PCR for gene of interest → gel extraction for gene of interest → gibson cloning → DH5a cells.
  • We are engineering our own plasmid to add to DH5a cells.
  • Store-bought plasmid (no gene of interest) cut it open → linearized plasmid (no gene of interest) (Gibson cloning) → homemade plasmid with gene of interest.
  • Glycerol stock (E. coli #1).
  • Plasmid backbone.
  • DH5a (E. coli #2).
  • Linearized backbone + gene of interest as DNA.
  • BL21 (E. coli #3).
  • Linearized backbone + gene of interest as protein.
  • DH5a (DNA) → miniprep → homemade plasmid (DNA) → BL21 (DNA) → central dogma of homemade plasmid (DNA A=T G=C) → transcription to RNA (A=U, G=C) → translation to protein (amino acids) → isolate human alpha-Amylase.

Lab 9
Lab 10

July 25, 2023

Attendees: Damini
Time present: 12:30 - 3:15
Gel Electrophoresis:
  • Create a 1x TAE dilution
    • 10% 10X TAE + 90% MQH2O
    • 100 mL 10X TAE + 900 mL water = 1000 ml (1L) 1X TAE
    • Add 100 mL of TAE and 900 mL of water to make the dilution
  • Make agarose gel
    • 1% gel = 1% agarose + 100 mL fresh TAE + 5 uL SYBR dye
    • 1g agarose + 100 mL fresh TAE
    • Microwave 45s → swirl → Microwave 45s → + 5 uL SYBR dye → pour in cold room → wait until solidified
  • Run gel 150 mV —> 45 min
    • 5 uL 6X loading dye + 25 uL pcr reaction → load 30 uL per well
    • Load 15 uL of ladder in the first (farthest left) well
    • DNA. d w d water
    • 1 2 3 7 8
Gel Extraction:
  • [Your gel extraction steps go here]

July 26, 2023

Attendees: Justin Hong, Heewon Choi
Creating an Overnight Culture (from Addgene):
  • Add KAN to LB broth (culture media) 100 μl 50 mg/mL stock to 100 mL LB broth (Steven’s email). Using these ratios, we combined 900 μl KAN:900 ml LB Broth (this is what we have). Then we inverted and relabeled LB Broth
  • Moved the solution of KAN and LB Broth to the bacteria room. Took bacterial culture out of the 37° C incubator. Added 50 mL of LB KAN to each erlenmeyer flask. Gently scraped bacteria from plate using pipette tip, and added one to each erylenmeyer flask. Added a foil cap and a label to each erlenmeyer flask, and placed each of the flasks to the shaking incubator at 37°C. Sealed the plates containing the bacterial culture and placed them in 4°C.

Lab 11

July 27, 2023

Attendees: Grace Lee, Damini Rosenblatt
  • Pour 45 ml of water into one conical tube for balance.
  • Pour 45 ml of bacteria in another tube and pour 50 ml of bacteria into two other tubes.
  • Put into a centrifuge to separate bacteria from growth media.
  • All the bacteria that was mixed with the growth media was separated to the bottom of the conical tube.
  • Pour out growth media opposite of the slope of the bacteria so it doesn’t get poured out.
  • Put bacteria into the freezer.
  • Pour 40 ml of ethanol to 2 Buffer PE Wash buffer 10ml concentrate.
  • Add 3 volumes of Buffer QG to 1 volume gel (100 mg gel ~100 pl). We have 726 mg of gel and multiply by 3 to get 2,178 volume (we used roughly 2,000 because tubes were too small).
  • Put gel with buffer QG into the incubator. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 min to help dissolve gel.
  • After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
  • Add 1 gel volume isopropanol to the sample and mix.
  • Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold.
  • To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Remove flow-through and place the QIAquick column back into the same tube.
  • Put 750 μl buffer PE into QIAquick and centrifuge for a minute or so to clean. Remove the flow through and place QiAquick back into the same tube.
  • Place the QIAquick column into a clean 1.5 ml microcentrifuge tube.
  • Remove the DNA by adding 50 μl water to the center of the QIAquick membrane and centrifuge the column for 1 min.
Lab 12
Lab 13
Lab 14

July 28, 2023

Attendees: Heewon, Daniel, Justin
Concentration of our plasmid from our miniprep:

Lab 15

August 1, 2023

Attendees: Heewon, Grace
Gel Electrophoresis:
  • Damini already made the TAE
  • Make agarose gel (original instructions)
    • 1% gel = 1% agarose + 100 mL fresh TAE + 5 uL SYBR dye
    • 1g agarose + 100 mL fresh TAE
    • Microwave 45s → swirl → Microwave 45s → + 5 uL SYBR dye → pour in cold room → wait until solidified
    • We are going to use 75 mL fresh TAE instead
  • Run gel 150 mV —> 45 min
    • 5 uL 6X loading dye + 25 uL pcr reaction → load 30uL per well
    • load 15 uL of ladder in the first (farthest left) well
  • DNA. d w d water
  • 1 2 3 7 8

Lab 16
Lab 17

August 2, 2023

Attendees: Heewon, Justin
Restriction digest
  • *All bands @ 6000nt/6kb
  • 6kb = plasmid
  • 5.5kb = broken/cut/linearized plasmid
  • ***MISSING .5 kb product
  • .5kb = excised MCS

We know the Restriction Digest failed because in the image of the gel on 8/1/23, there are no signs of the 5.5kb product or the .5kb product meaning the restriction enzymes did not work.

  • One thing we can try is to test different cut sites to make sure the manufacturer didn’t make any errors.
  • We will be trying:
    • Nde I(146bp)
    • Hind III(675bp)
  • We would expect a product of 529 bp and 5302 bp
  • Mix + match enzymes
  • Vary the amount of plasmid we’re asking to cut
    • Is there too much DNA for the enzymes to cut
    • We can try this today
Lab 20

August 3, 2023

Attendees: Damini
Finish gel extraction:
  • Placed gel on eyebright and take photo
  • Read ladder then cut out bands containing bacteria without cloning site
  • Add 3 volumes buffer to 1 volume gel
  • 350mg 652 mg* 411mg 340mg 384mg 324mg Total volume: 1809
    1050uL (1000 + 50) 1956uL 1233uL (1000 + 233) 1020uL (1000 + 20) 1152uL (1000 + 152) 972uL (972)
    1400 2608 1644 1360 1536 1296 7236 9045/800=11
    • *sample too large for column, eliminating from gel extraction. Put in fridge
  • Combine into 1 large tube
  • Pipette 800uLinto the spin columns
  • Place in centrifuge for 1 minute at top speed
  • Separate spin column from collection tube and discard flowthrough
  • Place in centrifuge and spin 1 min top speed again for residual flowthrough
  • 750 uL buffer PE
  • Repeat steps h and I
  • Separate spin column from test tube and placed in clean tube
  • Pipette 30 uL of buffer TE on each spin column membrane and spun in centrifuge
  • Discard spin column and combined all samples into a single sample
  • Nano Dropped to find concentration: 4.9 ng/uL
  • Place in yellow box in freezer

August 4, 2023

Attendees: Justin

Lab 21
Lab 22
Lab 23
Lab 24

August 9, 2023

Attendees: Justin

Lab 25
Lab 26
Lab 27
Lab 28

August 10, 2023

Attendees: Heewon, Justin

Lab 29
Lab 30

August 11, 2023

Attendees: Damini, Steven
Student Time Present: 1:30PM to 3PM
  • Make a 1% gel (8-1)
  • Do NEB restriction digest.
  • Using modifications follow procedure (8-1)
    • 3 rxns (1 uLDNA, 37*C, 30 min ( between 15-60 min))
      • Uncut control, 0 enzymes, same master mix
      • Xho 1 only = 1 enzyme, Xho 1
      • Xba 1 only = 1 enzyme, Xba 1
  • 1 Rxn;
    • H2O:36.6 uL
    • Buffer:5uL
    • Enzyme 1: 1uL
    • Enzyme 2: 1uL
    • 1 ug DNA: 6.4 uL

    • 0 enzymes —> H2O: 38.6 uL (+2) E1: 0 (-1) E2: 0 (-1)
    • 1 enzyme —> H2O: 37.6 uL (+1) E1: 1 uL E2: 0 (-1)
    • 1 enzyme—> H2O: 37.6 uL (+1) E1: 0 (-1) E2: 1 uL
    • 2 enzymes —> H2O: 36.6 uL E1: 1 uL E2: 1 uL
  • Protocols
    • 1 rxn 3.5 rxn
      H2O 38.6 uL 135.1 uL
      Buffer 5 uL 17.5 uL
      Xho 1 0 0
      Xba 1 0 0
      DNA 6.4 uL
      Total 50 uL (43.6 uL mix + 6.4 uL DNA per reaction)
    • 1 rxn 3.5 rxn
      H2O 37.6 uL 131.1 uL (limit by pip)
      Buffer 5 uL 17.5 uL
      Xho 1 1 uL 1 uL
      Xba 1 0 0
      DNA 6.4 uL
      Total 50 uL (43.6 uL mix + 6.4 uL DNA per reaction)
    • 1 rxn 3.5 rxn
      H2O 37.6 uL 131.3 uL
      Buffer 5 uL 17.5 uL
      Xho 1 0 0
      Xba 1 1 uL 1 uL
      DNA 6.4 uL
      Total 50 uL (43.6 uL mix + 6.4 uL DNA per reaction)
    • 1 rxn 3.5 rxn
      H2O 36.6 uL 128.2 uL
      Buffer 5 uL 17.5 uL
      Xho 1 1 uL 1 uL
      Xba 1 1 uL 1 uL
      DNA 6.4 uL
      Total 50 uL (43.6 uL mix + 6.4 uL DNA per reaction)
Reasoning for reaction:
  • Goal: express human alpha amylase protein (HAA)
    • To do that we need to put HAA in plasmid
    • To do that we need to cut out the multiple cloning site (MCS) using xho 1 and Xba 1 to cut out MCS then insert the gene of interest (GOI) to create the new plasmid
  • Our problem: the MCS won't get out of the plasmid
    • Is it the DNA?
      • Manufacturer: solutions- sequencing or PCR(shorter)
      • Concentration
    • Is it the enzymes?
      • If enzymes worked: we get MCS (~650bp) and plasmid (~6000 LP); not currently happening
      • Both enzymes

August 14, 2023 (Biology Team Orientation)

Attendees: Benjamin Li, Charlotte
Time Present: 9:30-11:30 AM, 1-4 PM

Learned lab safety procedures, took a tour of the lab

August 22, 2023

Attendees: Daniel, Steven
Student Time Present: 12PM to 3:45PM
  • Discussed the progress of our project.
  • We failed to cut our plasmid at 6650 bp (base-pairs) 3 times. The restriction enzymes are supposed to cut the plasmid, but NEB responded declaring that their enzymes were not the source of the problem. Thus, we wondered if the issue is with the plasmid.
  • OUR OBJECTIVE: To determine if the issue in cutting lies with the plasmid.
    1. Contact Addgene, who designed our plasmid
    2. Experiment: Sequence the plasmid
  • Began to take lab notes in a notebook Steven provided the team with. Daniel took handwritten notes.
  • Discussed the concept of sequencing: A technique used to determine the primary structure of an unbranched polymer.
  • In our project, sequencing will determine the order of bases/nucleotides of our plasmid.
  • Ordered a shipment to plasmidsaurus in order to check whether there is an issue with the plasmid.

Lab 31
Daniel taking notes
Lab 32
Daniel with the readied shipment to be made to plasmidsaurus for determining whether there is an issue with the plasmid.
Lab 33
Lab 34

August 23, 2023

Attendees: Hee won, Steven
Student Time Present: 6PM to 8PM
Sequencing of Plasmid
  • Plasmidsaurus was unable to sequence the plasmid, suggested DnAse based degradation.
  • Spoke with Ozge → plan is to make new mini prep of plasmid.
  • Check if the incubator can do 2 mL overnight cultures → color rack on black rubber mat @ 37 Celsius.
  • 8 x 2 mL overnight culture.

August 29, 2023

Attendees: Daniel, Steven
Student Time Present: 7PM to 7:30PM
Overnight Culture

8 x 2 mL @ 37 Celsius shaker.

August 30, 2023

Attendees: Hee won, Steven
Student Time Present: 7PM to 7:30PM
Overnight Culture

8 x 2 mL → 6X frozen pellets @ -20 Celsius.

September 5, 2023

Attendees: Justin, Rin, Steven
Student Time Present: 4PM to 8:30PM
  • New Qiagen kit → 24 mL 100% ELDH.
  • 6X pellet → 300 uL @ 44.5 ng / uL → @ -20 Celsius.
  • → kit suggested 2 ug / ~ 2mL culture, yield seems correct (44 x 50 x 6 = 13,200 ng).
  • Could this have worked?
  • → put two old samples to the side
  • - will do digest @15 min w/ 1 ug DNA (22.5 uL plasmid)
  • - do A and D from 08/11/2023 (DR)

September 7, 2023

Attendees: Hee won, Justin, Daniel, Steven
Student Time Present: 6PM to 8:45PM
Restriction Digest

Following 08-11 protocol for sections and A and D.

September 14, 2023

Attendees: Hee won, Steven
Student Time Present: 6PM to 8:30PM
Checking the Bacteria

Current bacteria grows on Kanamycin (KAN) plates BUT does NOT get cut by XbaI and XhoI.

Is this the bacteria that we really want?

Growing the bacteria (from addgene 135521) on empty plates, KAN plates (single line), AMP plates (double line).

September 16, 2023

Attendees: Daniel, Hee won, Steven
Student Time Present: 7PM to 8PM
Agar Plates

Made AMP Agar plates → two stripes.

September 17, 2023

Attendees: Daniel, Steven
Student Time Present: 7PM to 8PM
Streaking Plates

Streaking plates with NEW stab of backbone

September 21, 2023

Attendees: Justin, Hee won, Steven
Student Time Present: 7PM to 7:45PM
Overnight Cultures

Overnight cultures 6*3mL

September 22, 2023

Attendees: Daniel, Justin, Steven
Student Time Present: 6PM to 8:30PM
Mini-prep for Plasmid Backbone

Followed protocol from 08-11. Generated 15.3 ng/uL in about 400 uL.

September 28, 2023

Attendees: Daniel, Justin, Steven
Student Time Present: 6PM to 8:30PM
Restriction Digest

Follow the protocol from 09-07. Determine 1 µg of DNA.