Transformation of plasmids

1. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins.)
2. Remove agar plates (containing the appropriate antibiotics) from storage at 4°C
   • Let warm up to room temperature, then
   • (optional) incubate in 37°C incubator.
3. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 μL of competent cells in a microcentrifuge. GENTLY mix by flicking the bottom of the tube with your finger a few times.
4. Incubate the competent cell/DNA mixture on ice for 20-30 mins
5. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 45secs
6. Put the tubes back on ice for 2 mins.
7. Add 250-1,000 μl LB or SOC media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45min

Materials

• Ice bucket filled with ice
• Microcentrifuge tubes
• Sterile spreading device
• Agar plate
• 42°C water bath
• Shaking incubator
• DH5alpha (Or any other possible bacteria)
• (Plasmid #17402)
• LB or SOC medium
• Kanamycin,50 μg/mL
• LB agar plate (with Kanamycin)
• Stationary incubator at 37 °C

Selection

1. Plate some or all of the transformation onto a 10 cm LB agar plate containing the Kanamycin. Use swab in "Z" motion to spread bacteria evenly.
2. Incubate plates at 37°C overnight.
3. Only DH5alpha with succesful transfomation of plasmids will be able to grow and divide into colonies.

Materials

• Kanamycin,50 μg/mL
• LB agar plate (with Kanamycin)
• Stationary incubator at 37 °C

Plasmid Extraction

1. Before starting: add 24 ml of ethanol to 6 ml of Monarch Plasmid Wash Buffer 2

   Note: All Incubation are under 35"C

   All centrifugation steps should be carried out at 16,000 x g (-13,000 RPM)

   Store Plasmid Neutralization Buffer (83) at 4"C after opening

2. Pellet 1.5 ml of culture bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant. Ensure cultures are not overgrown (12-16 hours is ideal).
3. Resuspend pellet in 200 ul Plasmid Resuspension Buffer (B1) Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
4. Lyse cells by adding 200 ul Plasmid Lysis Buffer (B2).
5. Invert tube immediately and gently 5-6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.
6. Neutralize the lysate by adding 400 Ul of Plasmid Neutralization Buffer (B3) Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
7. Clarify the lysate by spinning for 3 minutes at 16,000 x g
8. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
9. Re-insert column in the collection tube and add 200 ul of Plasmid Wash Buffer 1.
10. incubate for 5 minutes, then Centrifuge for 1 minute. Discard the flow-through
11. Add 400 pl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
12. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through.
13. Add 30 Ul DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA

Materials

• ethanol
• Monarch@ Plasmid Miniprep Kit
  • Monarch. Plasmid Miniprep Columns
  • Monarch. DNA Elution Buffer
  • Monarch. Plasmid Wash Buffer 1
  • Monarch. Plasmid Wash Bufter 2
  • Monarch. Plasmid Resuspension Buffer (81)
  • Monarch. Plasmid Lysis Buffer (B,2)
  • Monarch. Plasmid Neutralization Buffer (83)
• pipette
• pipette tip
• microfuge tube
• incubator
• centrifuge

Plasmid Extraction

1. Before starting: add 24 ml of ethanol to 6 ml of Monarch Plasmid Wash Buffer 2

   Note: All Incubation are under 35"C

   All centrifugation steps should be carried out at 16,000 x g (-13,000 RPM)

   Store Plasmid Neutralization Buffer (83) at 4"C after opening

2. Pellet 1.5 ml of culture bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant. Ensure cultures are not overgrown (12-16 hours is ideal).
3. Resuspend pellet in 200 ul Plasmid Resuspension Buffer (B1) Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
4. Lyse cells by adding 200 ul Plasmid Lysis Buffer (B2).
5. Invert tube immediately and gently 5-6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.
6. Neutralize the lysate by adding 400 Ul of Plasmid Neutralization Buffer (B3) Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
7. Clarify the lysate by spinning for 3 minutes at 16,000 x g
8. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
9. Re-insert column in the collection tube and add 200 ul of Plasmid Wash Buffer 1.
10. incubate for 5 minutes, then Centrifuge for 1 minute. Discard the flow-through
11. Add 400 pl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
12. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through.
13. Add 30 Ul DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA

Materials

• Chemicals required:
  • 1 µg DNA
  • 0.5 µL of Restriction Enzyme(s)
  • 10x rCutsmart Buffer
  • 10-50 µL distilled water
• Instruments required:
  • pipette
  • pipette tip
  • ice bucket
  • microfuge tube
• Apparatus required:
  • incubator

Setting up an agarose gel

1. For a small gel, add 20 ml 1xTAE buffer to a conical flask. (If there is none, dilute the 50xTAE buffer by 50 times.)
2. Then, add 0.2 g agarose (1%) to the conical flask and heat it by microwave oven by 30-45 s to dissolve it until it becomes a clear and transparent liquid.
3. Cool it down a little bit by running water for around 15 s.
4. Add about 1 μl (for 20 ml TAE) of DNA staining dye, red safe (20000x).
5. Pour the solution to the white tightened tank with gates to allow it to solidify. Add the gel comb so as to create wells for the gel. Wait >15-30 min until it is gel-like and ready to use.

Running agarose gel

6. Orient the gel with wells (comb removed) facing the BLACK negative electrode. Check if the gel is covered by TAE buffer in the tank.
7. Add 6x/10x loading dye to the DNA to a total volume of 25 µl (or less) before adding to the wells. Mix loading dye to DNA to make the solution colored.
8. C. Load the sample to the wells (25 µl (or less)/ well)
9. Add 3-5 µl DNA ladder to a separate well.
10. Connect the electrodes to the power supply with correct color, black to black, red to red. Apply power supply with 120 V. Check if there are bubbles on the negative electrodes.
11. Allow it to run for about 30 mins
12. After electrophoresis for 30 min, disconnect power, take the gel to imager, and turn UV on to observe bands.Never run a gel with >200V, as the heat so generated can melt the gel and also easier to cause electric leakage. Range from 80 – 160 V is acceptable. Usually it needs at least 100 ng DNA for a band to be seen and visualized on the UV trans-illuminator

Materials

• Chemicals required:
  • 1 µg DNA
  • 0.5 µL of Restriction Enzyme(s)
  • 10x rCutsmart Buffer
  • 10-50 µL distilled water
• Instruments required:
  • pipette
  • pipette tip
  • ice bucket
  • microfuge tube
• Apparatus required:
  • incubator

Purifying DNA Protocol

1. Once you have run your gel, move it to an open UV box (be sure to wear proper UV protection!), remove it from any gel tray.
2. Place the gel in a labeled microfuge tube
3. Add 1 ml Binding Buffer to the gel slice
4. Incubate the gel mixture at 50-60 °C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to faciliate the melting process.
5. Vortex the gel mixture briefly to ensure that the gel is completely dissolved
6. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 μL of 3M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow
7. Transfer up to 800 μL of the solubilized gel solution (from step 3 or 4)to the purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube
8. Add 700 μL of Wash buffer to the GeneJET purification column. Centrifuge for 1min. Discard the flow-through and place the column back into the same collection tube
9. Centrifuge the GeneJET purification column for an additional 1 min to completely remove residual wash buffer
10. Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge tube with pipette. Add 50 μL of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 min
11. Discard the GeneET purification column and store the purified DNA -20°C

Materials

• 100ml Ethanol 96%-100%
• 10μL 3 M sodium acetate, pH 5.2
• GeneJET Gel Extraction Kit
  • 1 mL Binding buffer
  • 700μL wash buffer (concentrated)(45ml)
  • 50μL Elution Buffer
  • GeneJET Purification Columns
• Instruments required:
  • pipette
  • pipette tip
  • DNA cleanup columns (5 μg)
  • Collection Tubes (2ml)
  • 1.5 ml microfuge tube
• Apparatus required:
  • Centrifuge

Purifying DNA Protocol

1. Thaw and resuspend T4 DNA ligase buffer at room temperature

2. Set up the following reaction in a microcentrifuge tube on ice.

   T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes. Use NEBioCalculator to calculate molar ratios.

   COMPONENT	20 μl REACTION
   T4 DNA Ligase Buffer (10X)	2 μl
   Vector DNA (4 kb)	50 ng (0.020 pmol)
   Insert DNA (1 kb)	37.5 ng (0.060 pmol)
   Nuclease-free water	to 20 μl
   T4 DNA Ligase	1 μl

3. Gently mix the reaction by pipetting up and down and microfuge
4. For sticky ends, incubate at 16°C overnight (or room temperature for 10 minutes).
5. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
6. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 μL of 3M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow
7. Heat inactivate at 65°C for 10 minutes.
8. Cool on 0°C ice and transform 1-5 μl of the reaction into 50 μl competent cells.

Materials

• T4 DNA Ligase Buffer (10X)*
• vector DNA
• Insert DNA
• Nuclease-free water
• T4 DNA ligase

Purifying DNA Protocol

Colony PCR

1. Dispense PCR master mix (20 µL) into each PCR tube or plate.
2. Using a sterile micropipette tip or a sterile toothpick transfer cells from each colony to a PCR tube and briefly stir to resuspend them in the PCR master mix. The mix may look slightly cloudy. Don’t pick too many cells. Overloading cells will interfere with the PCR.
3. Amplify target DNA with the following thermocycling conditions

   Step	Temperature	Time	Cycles
   Cell lysis	94 °C	5 to 10 minutes	1
   Denaturation	0.5 µL	0.5 minutes	30-40
   Annealing	45 to 68 °C	0.5 minutes
   Extension	72 °C	1-2 minutes(~ 1 kb/min)
   Hold	4 °C	Indefinitely

4. After PCR, load 5 to 10 µl of each reaction directly into a 1% agarose gel, allowing a lane for an appropriate ladder, and perform electrophoresis.

Materials

• REDExtract-N-Amp™ PCR ReadyMix™ or JumpStart™ REDTaq® PCR ReadyMix™
• Forward Primer (20 µM)
• Reverse Primer (20 µM)
• Water

Conjugation of PEG to PAMAM Dendrimers

1. The PEG with two functional groups (NHS-PEG-MAL) was diluted in phosphate-buffered saline (PBS; pH 8.0) to 10 mg/mL
2. The PAMAM and NHS-PEG-MAL (molar ratio of 1:2) were stirred at room temperature for 2 h in the dark.
3. The resulting mixture was concentrated twice by an ultrafiltration device (molecular weight cut-off: 10 kDa) at 12,000 rpm for 20 min at 4°C
4. Resolubilize resulting mixture in PBS (pH 7.0). The final product was lyophilized to obtain PAMAM-PEG-MAL.

Materials

• Maleimide PEG3500 succinimidyl carboxymethyl ester (NHS-PEG-MAL, MW 3500)
• Lyophilized PAMAM G5 dentrimer
• phosphate-buffered saline (PBS; pH 8.0)
• phosphate-buffered saline (PBS; pH 7.0)
• ultrafiltration device (molecular weight cut-off: 10 kDa)

Preparation of 3'-SH-APT

1. 0.1 µmol OH-(CH2 )6 -S-S-(CH2 )6 -APT was dissolved in 2.5 mL 100 mM DTT (pH 8.0)
2. incubated at room temperature for 30 minutes
3. The entire sample was loaded onto a Glen Gel-Pak™ 2.5 (Glen Research, Sterling, VA) desalting column, equilibrated with 25 mL 50 mM sodium phosphate (pH 6.0), allowed to drip through
4. elute sample with 2.5 mL sodium phosphate (pH 6.0)
5. Collect the purified conjugation of SH-APT.

Conjugation of aptamer and PAMAM

6. 0.2 µmol MAL-activated PAMAM-PEG was dissolved in 3 mL nuclease-free phosphate-buffered solution (pH 7.0), reacted with 0.1 µmol SH-APT for 12 hours at 4°C,
7. the resulting product, PAMAM-PEG-APT, was purified by ultrafiltration using a 20 kDa molecular weight cutoff membrane to remove unreacted aptamer
8. suspend the purified PAMAM-PEG-APT in nuclease-free water.
9. The anti-PSMA aptamer-conjugated PAMAM-PEG (PAMAM-PEG-APT) was stored at 4°C.

Ultrafiltration

10. Essentially using membrane to perform filtration. Same procedures as the filtration but not cone shaped! Best recommended using : https://youtu.be/a0PpQlxiDt0?si=MC9KiKi909_Q9v32

source

11. https://www.dovepress.com/second-generation-aptamer-conjugated-psma-targeted-delivery-system-for-peerreviewed-fulltext-article-IJN

Materials

• Synthesized PAMAM-PEG
• dithiothreitol (DTT) (pH 8.0)
• anti-PSMA aptamer (A10-3.2)
• nuclease-free water
• sodium phosphate (pH 6.0)
• 20 kDa molecular weight cutoff membrane
• Glen Gel-Pak™ 2.5 desalting column
• Incubator

1. Mix BODIPY-493/503 NHS ester and PAMAM-PEG-EpDT3 in a molar ratio of 5:1 in NaHCO3 working solution (100mM). Stir the solution at 4°C in the dark for 12 hours
2. Remove the unreacted BODIPY by using an ultrafiltration device, which works at 12000 rpm for 20 minute at 4°C
3. The end-product was collected and lyophilized
4. Add PAMAM-PEG-APT into cell line, observe dyeing under fluorescence microscope

Materials

• BODIPY-493/503 NHS ester
• PAMAM-PEG-APT (polymer w/ PEG + aptamers)
• NaHCO3 working solution
• ultrafiltration device (MWCO 10,000)

1. Dilute plasmid to 100 µg/mL with 50 mM Na2SO4
2. Mix the mixture with newly synthesized PAMAM-PEG-APT at different nitrogen-to-phosphorus (N/P) ratios for 30 s and incubated at room temperature for 30 min.Try with different PAMAM-PEG-APT concentrations.
3. Perform gel electrophoresis to see the minimum N/P ratio for no DNA to be observed
   • If all DNA is combined into the polymer, they will not move up the agarose gel.
   • Use naked plasmid as a control
4. (Use centrifuge to separate the polymer that has not taken up plasmid)
   • polymer without plasmid should be lighter and can be discarded as supernatant

N-P Ratio

5. https://www.mirusbio.com/n-to-p-ratio/
   • 3×10-9 mol of P / 1 µg of nucleic acid

Materials

• PAMAM-PEG-APT conjugate
• 50 mM Na2SO4
• Plasmid

A. Material Preparation

1. For Working Solution (100 reactions), add 50µL of 100X Coelenterazine to 5mL of Gaussia Flash Assay Buffer. Use 50µL of the Working Solution per reaction.
2. For 1X Cell Lysis Buffer, dilute 2X Cell Lysis Buffer with an equal volume of ultrapure water.
3. For BupH Modified Dulbecco's PBS Packs, they are pouches of dry-blend powder that make physiological sodium- and potassium-phosphate buffered saline (D-PBS) for ELISA and western blot diluents and wash buffers. Each pack makes 8 mM sodium phosphate, 2 mM potassium phosphate, 0.14 M NaCl, 10 mM KCl modified Dulbecco's PBS buffer at pH 7.4 when dissolved in 500 mL of water.

B. Cell Transfection (Should be completed with other methods)

4. Plate ~10,000 cells/well in a 96-well plate. Incubate plates overnight at 37°C in 5% CO 2 . If using a different plate size, adjust the cell number accordingly. Use only cells growing in log phase at a passage number ≤ 15.
5. Use a standard protocol to transfect mammalian cells with a Gaussia luciferase plasmid.
6. Incubate cells for 16-72 hours at 37°C in 5% CO 2 in a cell culture incubator.
7. Proceed with the individual experimental protocol for cell treatment

C. Collection of Media and Cell Lysis

8. Remove 10-20µL of media from the transfected cells within 72 hours after transfection.
9. Lyse cells to monitor intracellular luciferase activity. Rinse the cells with 100µL/well of 1X DPBS buffer, aspirate DPBS and add 50-100µL/well of 1X Cell Lysis Buffer. Do not disturb the cell monolayer during the transfer and wash steps
10. Rotate the plate on a platform shaker at moderate speed for 15 minutes. Check for complete cell lysis using a light microscope. If lysis is incomplete, continue shaking the plate for an additional 15 minutes.

D. Gaussia Luciferase Flash Assay

11. Add 10-20µL/well of cell lysate or media to a white or black, opaque 96-well plate.
12. Program the plate reader; if using an injector, prime the injector with Working Solution.
13. Add 50µL of Working Solution to each well.
14. Immediately after adding the reagent, detect the light output.

Materials

• Modified Dulbecco’s Phosphate-buffered saline (DPBS)
• Pierce™ Gaussia Luciferase Flash Assay Kit (From ThermoFisher)
  • Gaussia Flash Assay Buffer, 5mL, store at 4°C
  • Coelenterazine (100X), 50µL, store at -80°C
  • 2X Cell Lysis Buffer, 6mL, store at room temperature