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Design-Build-Test-Learn cycle
Design: We derived our backbone from the pICH41308 plasmid from the MoClo Toolkit [1][2]. pICH41308 is a Level 0 cloning vector with a length of 2247 base pairs that contains LacZ alpha and an antibiotic resistance to spectinomycin. At first, the experiment was conducted using un-optimized inserts; we later transitioned to using codon-optimized inserts. All of these inserts are considered Level -1 parts and contain BbsI restriction enzyme sites.
Build In order to integrate these inserts into the MoClo backbone, we utilized Golden Gate Assembly. Golden Gate Assembly (GGA) is the method by which one or more inserts can be efficiently integrated into a vector backbone by using a Type IIS restriction enzyme and a T4 DNA ligase in a one-pot reaction. The Type IIS restriction enzyme in particular has the ability to cleave DNA outside of its recognition sites, which ultimately results in the enzyme's removal of the recognition sequence. This method is advantageous because of the fact that scar sequences are not introduced, multiple fragments can be integrated into the backbone, and the digestion and ligation processes can be performed simultaneously.
Test: The efficacy of our GGA reaction was tested through multiple steps: electroporation and touchdown PCR. We initially used electroporation in order to transform our assembled plasmid into electrocompetent E.coli Top10 cells. Following electroporation, we used X-Gal plates to do blue-white screening for transformed colonies. The idea was that a colony would turn blue if it still contained the LacZ fragment in its genome; if a colony was blue, that would indicate that it was not transformed with the plasmid due to the fact that the insert should, hypothetically, knock out the LacZ fragment. If a colony turned white, that would indicate that the colony did not contain the LacZ fragment and was hence transformed with the plasmid.
Learn: Following the transformation of Top10 E.coli cells, we performed touchdown PCR to see if our GGA reaction worked by confirming the size of our parts, run with a subsequent gel electrophoresis. After testing our GGA, we came to the consensus that our reaction was unsuccessful, but we decided to troubleshoot the testing steps first. The first conclusion we came to was that electroporation was unsuccessful due to the fact that the restriction enzymes from the GGA led to high salt concentrations that would cause the E.coli cells to be unable to uptake the plasmid; this led us to switch to chemi-competent transformation.
Design: As mentioned before, the high salt concentration led us to switch to chemi-competent transformation. Another area of concern was our PCR protocol: previously, we had been following the Q5 protocol[9] using colonies from the transformed plate, whereas we should have been following the OneTaq protocol[10] where we were doing colony PCR. Another limitation we had realized was regarding the blue-white screening process, which could have been potentially unreliable due to the fact that we were almost exclusively getting white (transformed) colonies. Due to this, we decided to pick ten individual colonies and screen all of them during our PCR process; this was running on the assumption that only some of the colonies were truly transformed with our level 0 part.
Build and Test: After troubleshooting all of the testing steps following our GGA reaction, we ran a gel electrophoresis. A successful GGA reaction was indicated by an absence of a band around 300 base pairs; since that is the size of the region between the flanking primers in lieu of the LacZ fragment. The gel that we ran showed a band around 300 base pairs, which suggested an unsuccessful GGA reaction due to the fact that the LacZ fragment was removed from the backbone, but the insert was not integrated into the vector.
Build and Test: After troubleshooting all of the testing steps following our GGA reaction, we ran a gel electrophoresis. A successful GGA reaction was indicated by an absence of a band around 300 base pairs; since that is the size of the region between the flanking primers in lieu of the LacZ fragment. The gel that we ran showed a band around 300 base pairs, which suggested an unsuccessful GGA reaction due to the fact that the LacZ fragment was removed from the backbone, but the insert was not integrated into the vector.
Learn: From this, we concluded that our reagents could potentially be an impedance to us having a successful GGA reaction, and that our GGA reaction itself needed to undergo troubleshooting.
Design: In order to troubleshoot our GGA reaction and the reagents we were using, we set up a three-part control experiment with the following design:
Control 1: pICH41308 + GFP + T4 Ligase Buffer + DI H2O
Control 2: pICH41308 + GFP + T4 Ligase Buffer + Bbs1 Restriction Enzyme + DI H2O (no T4 Ligase)
Control 3: pICH41308 + GFP + T4 Ligase Buffer + Bbs1 Restriction Enzyme + T4 Ligase Buffer + DI H2O
Using this control experiment, (1) and (3) should hypothetically work when plated; (1) was the plasmid and (3) was the full integrated plasmid.
(2) should not have worked because it only contained digested plasmid that had not been ligated using the T4 Ligase.
Build: After running the troubleshooting experiment, it was concluded that the Bbs1 restriction enzyme stock we were using was defective. After running a GGA reaction, all three parts of the experiment were plated on X-Gal + Spectinomycin after undergoing a chemi-competent transformation into DH5-alpha E.coli cells, after discovering that Top10 cells inherently contained a resistance to spectinomycin.
Test: The plate for (1) was successful, as it only contained the untransformed backbone; the plates for (2) and (3) were unsuccessful as the digestion step was not able to occur due to the ineffectiveness of the Bbs1 restriction enzyme.
Learn: From here, we were able to pinpoint the troubleshooting areas for Golden Gate, and could now have the potential to successfully create a level 0 part, with the goal of eventually being able to create a level T construct.
Design: After much troubleshooting with the GGA itself and every step of the testing process, we were able to develop a steady pipeline for being able to develop level 0 parts with the goal of eventually creating a level T construct.
Build: The final design are as listed below:
GGA: integrate optimized level -1 inserts with Bbs1 restriction sites into pICH41308 MoClo backbone
Transformation: transform chemi-competent DH5-alpha cells with GGA product; plate and incubate overnight using blue-white screening.
PCR: pick ten different transformed white colonies from plates and dilute in 10 μL DI H2O; follow the NEB protocol using OneTaq 2X MasterMix,
adjust annealing temperature and extension time in accordance with the insert present in the amplicon.
Gel: run the PCR samples on a 1% agarose gel and observe the size of the bands.
Test: With this new pipeline in mind, we tested using codon-optimized inserts of Cpf1, GFP, CbAgo, and more (for all of the parts, look at the Contribution page!). Throughout our testing process, we were able to prove that we had successful GGA reactions for codon-optimized Cpf1, GFP, and CbAgo. In addition to having these level 0 reactions, we were also able to ligate a level 1 antibiotic-resistance construct with success using the appropriate parts from the CyanoGate Toolkit.[8]
Learn: In order to confirm the identity of the level 0 parts that were isolated, we sent many of our samples to UC Berkeley for sequencing.
Protocols
The following is the most up-to-date set of protocols we implemented throughout the project. Each protocol section contains 3 sections: Materials, Protocol, and Troubleshooting Tips. It's important to highlight that many of these protocols were specifically designed for troubleshooting purposes. Some were developed in response to challenges encountered during the project, while others were fine-tuned to ensure smooth operations. These protocols reflect the evolving nature of our approach and were essential in addressing and mitigating any unforeseen issues along the way.
Materials
- Sample DNA
- Zymo Zyppy Plasmid Miniprep Kit (Zymo Research D4036)
Protocol
Note: Make sure to time all of your steps where the timing matters!
- Spin down 5mL of culture in large centrifuge at 2000 rcf for 5 minutes
- Dump out supernatant, cut the pipet tip & resuspend pellet in 600uL of DI water
- Transfer sample to 1.5mL microcentrifuge tube
- To sample, add 100uL of 7X Lysis Buffer (blue) and mix by inverting the tube 4-6 times
- Proceed to step 5 within 2 minutes (opaque/clear blue = lysis indicator)
- Add 350uL of Cold Neutralization Buffer (yellow) and mix thoroughly. Sample will turn yellow when neutralization is complete and yellowish precipitation will form. Invert the sample an additional 2-3 times to ensure complete neutralization
- Wait until about 1 minute 50 seconds to add Neutralization buffer
- Only invert until the blue color goes away and no more
- Centrifuge at 11000-16000 rcf for 6 minutes in small centrifuge
- Transfer the supernatant (~800uL – don't be greedy!!) into the Zymo-Spin In column, avoid disturbing the cell debris pellet
- Place the column into a collection tube and centrifuge for 30 seconds
- Empty the collection tube after each spin down
- Discard the flow through and place the column back into the same collection tube
- Add 200uL of Endo-Wash Buffer to the column, centrifuge for 30 seconds
- Add 400uL of Membrane Wash Buffer to the column, centrifuge for 1 minute
- Repeat this step twice
- Then do one more spin down without anything in it
- Transfer the column into a clean 1.5mL microcentrifuge tube, then add 30uL DI water directly to the column and let it stand more 1 minute at room temperature
- Centrifuge for 30 seconds to elute the plasmid DNA
- Nanodrop to verify results
Troubleshooting Tips
- Do not spin too fast for the initial spin
- We spin at 2000 rcf for 6 mins
- Check reagents to ensure they are still good
- Choose single colonies
- Spin down in the 15 ml tubes
- We made our own membrane wash buffer
- Perform 2 membrane wash steps
- Spin 1 more time after membrane wash to get rid of any leftover ethanol
- MAKE SURE NEUTRALIZATION BUFFER IS PUT IN FRIDGE
Materials
- Sample DNA
- 100% EtOh
- 3M sodium acetate
- 75% EtOH
- MilliQ water
Protocol
- Starting with ~58μL sample
- Add 2.5-3.0 volumes ice cold 100% ethanol
- Add 0.1 volumes 3M sodium acetate
- Vortex to mix
- Precipitate overnight at -20℃
- Centrifuge at full speed (16000 rpm) for 30 minutes, at 4℃
- Wash pellet twice with 500μL ice cold 75% ethanol
- Spin at 4℃ for 10 minutes
- Take ethanol out, spin at full speed to remove trace amounts
- Air dry & resuspend in 25μL nuclease free water
Troubleshooting Tips
- Leave DNA mixed with sodium acetate and EtOH overnight for better results
- Make sure you have the hinges facing outwards when you centrifuge so that you know where the DNA will collect if a pellet is not present
Materials
- Sample DNA
- Zymo Quick DNA HMW Magbead Kit (Zymo Research D6060)
- 70% EtOH
- MilliQ Water
- Shaker
Protocol
Note: Do not pipet to resuspend !
- Add 400uL (equal volume) Quick-DNA MagBinding Buffer to 400uL of the sample
- Resuspend by flicking the tube or dragging it across the microcentrifuge tray
- Add the 33uL of MagBinding Beads to each sample
- Resuspend by flicking the tube or dragging it across the microcentrifuge tray and place the sample on a shaker for 10 minutes (speed 3-4)
- Transfer the sample to the magnetic stand until beads have separated from the solution, then remove & discard the supernatant, transfer the sample off of the magnetic stand
- Add 500uL Quick-DNA MagBinding Buffer
- Resuspend the beads by flicking the tube or dragging it across the microcentrifuge tray, then place the sample on shaker for 5 minutes
- Transfer the sample to the magnetic stand until the beads have separated from solution, then remove and discard the supernatant, transfer the sample off of the magnetic stand
- Add 500uL DNA Pre-Wash Buffer
- Resuspend the beads by flicking the tube or dragging it across the microcentrifuge tray
- Transfer the sample to the magnetic stand until the beads have separated from solution, then remove and discard the supernatant, transfer the sample off of the magnetic stand
- Add 900uL 70% EtOH & resuspend the beads by flicking the tube or dragging it across the microcentrifuge tray
- Transfer all liquid to a new microcentrifuge tube
- Transfer the sample to magnetic stand until beads separated from solution, remove and discard the supernatant, transfer sample off of magnetic strand
- Repeat steps 12-14
- To dry the beads, transfer the sample to 55℃ incubator & incubate for 10 minutes (or let air dry for 20 minutes)
- Add 50uL of DI water to each sample
- Put sample on shaker for 5 minutes at room temperature
- Transfer the sample to a magnetic stand until beads have separated from solution, then transfer the eluted DNA to a new tube
- Nanodrop
- Run a gel to verify results
Troubleshooting Tips
- Let samples sit in DNA/RNA shield for 1 hour at room temperature
- Add proteinase K and incubate for 2 hours at 55°C
- Add RNAse A and incubate at room temperature for 30 mins
- Instead of g-DNA wash buffer, use 70% EtOH
- NO PIPETTE MIXING
Materials
- Agarose powder
- 1xTBE
- Sample DNA
- Gel Dye
- Gel Box
- MilliQ Water
Protocol
- Measure 0.8g-2g of agarose depending on the fragment size intended to be resolved
- Mix agarose powder with 100 mL 1xTBE in a 200mL microwavable flask
- Microwave for 1-3 min until the agarose is completely dissolved
- Let the agarose solution cool down, add 5 ul of gel dye and cool to about 50 °C for about 5 mins
- Pour the agarose into a gel tray with the well comb in place
- Let newly poured gel sit at room temperature for 20-30 mins, until it has completely solidified
- Fill the gel box with 0.5x TBE until the gel is covered
- Carefully load a molecular weight ladder into the first lane of the gel
- Carefully load your samples into the additional wells of the gel
- Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel
- A typical run time is 1-1.5 hours, depending on the gel concentration and voltage
- Visualize results
Troubleshooting Tips
- Make sure you microwave the TBE and agarose until agarose is fully dissolved into the solution
- Microwave in intervals, we did 1 min, 20 sec., and then 10 sec until fully dissolved
- Wait around 15 sec. between microwave intervals, or the solution could boil over
- Use more loading dye to ensure your samples visualize on the gel
- We went from 1µL to 1.5 µL
- DO NOT FORGET TO ADD GEL STAIN
- And don't forget to put it back in the fridge when done
- Remember to use the same TBE buffer you use to make the gel to run the gel
Materials
- Thermocycler
- BbsI-HF or Bsa1-HFv2
- T4 DNA Ligase
- 10x T4 DNA Ligase Buffer
- Destination plasmid
- Insert
- MilliQ Water
Protocol
-
For each tube, prepare the following reaction:
Reagent Volume (μL) BbsI-HF or Bsa1-HFv2 1.5 T4 DNA Ligase 0.5 10x T4 DNA Ligase Buffer 2.5 Destination plasmid 1 Insert 0.75 per insert Milli Q H2O Up to 19.5 Total: 25 μL -
Place sample in the thermocycler with the following steps:
Step Temperature (℃) Time (min) 1 37 5 2 (30x cycles) 16 5 3 60 5 Hold 4 ∞ - If done overnight, hold the protocol at 4℃, then do step 3 the following day prior to transformation.
- Run a transformation with chemi-competent E. coli Top10 cells subsequent to Golden Gate Assembly
Troubleshooting Tips
- To verify golden gate products, design primers that target the ligation sites
- Increase the molar ratio of insert to backbone from 2:1 to 3:1 / 4:1 to ensure the inserts ligates into the backbone
- C1V1 = C2V2 is your best friend
- If your parts contain a LacZ fragment, it is helpful to make x-gal plates to test if your insert actually ligated to your backbone
Materials
- SOC Medium
- E. coli cells (previously made chemi-competent)
- Golden Gate Assembly Plasmid Product
- Hot Water Bath
- Incubator
- LB+X-Gal+IPTG+Spec Plates
Protocol
- Take competent cells out of -80℃ and thaw on ice until no longer frozen.
- Mix 1-5 μL of plasmid product into 50 μL of competent cells (aim for 10 ng of plasmid for 50 μL of competent cells). Resuspend using a pipet (cut the tip in order to avoid lysing the cells).
- Incubate the transformation tube on ice for 30 minutes.
- Heat shock the transformation tube by placing it in a hot water bath heated to 42 ℃ for 90 seconds.
- Immediately place the tube on ice for 1 minute.
- Add 800 μL of SOC medium immediately after as a recovery step.
- Place the tube in a shaking incubator for 45 minutes, under 37 ℃ and 225rpm.
- Take the liquid culture out of the incubator and plate using 25-50 μL of liquid culture. Incubate the plates overnight for 12-16 hours.
- Once taken out of the incubator, look for white colonies in order to select for those which are transformed (blue colonies indicate untransformed colonies).
Troubleshooting Tips
- Know the genotype of your competent cells
- For instance Top10 are naturally resistant to Streptomycin, so a Strep plate would not be good for selection
Materials
- Thermocycler
- OneTaq 2x Master Mix with Standard Buffer (NEB)
- X-Gal plates containing transformed E. coli colonies
- Primer (2.5 μM)
- Reverse Primer (2.5 μM)
- Template DNA
- MilliQ Water
Protocol
- Take plates with grown, transformed colonies and pick 1 colony per 25 μL PCR reaction.
-
Set up the following PCR reaction:
Reagents Volume (μL) Forward Primer (2.5 μM) 2 Reverse Primer (2.5 μM) 2 Transformed Colony Sample 1 OneTaq 2x Master Mix 12.5 MiliQ Water 7.5 Total: 25 μL -
Program the thermocycler with the following conditions:
Step Temperature (℃) Time (seconds) Denaturing 94 30 Annealing 51 45 Extension 68 1 min per kb Repeat the steps below for 20 cycles, while setting up a gradient for the annealing step.
Step Temperature (℃) Time (seconds) Denaturing 94 30 Annealing 48-53 45 Extension 68 1 min per kb Program the following steps for safety.
Step Temperature (℃) Time (minutes) Final Extension 68 5 Hold 4 ∞ - Perform a gel electrophoresis in order to visualize the results of the colony PCR
Troubleshooting Tips
- Make sure primers are diluted to correct molarity
- Usual molarity is 10 mM, but we adjusted to 2.5 mM to get a more accurate volume
- Make a master mix first containing your buffer and DNA polymerase
- Aliquot out your master mix to prevent freeze thaw cycles
- We had a final volume of 25 µL and we had 2x mastermix, so we aliquoted out 12.5 master mix
- Calculate out your primers annealing temp
- NEB Tm calculator is a big help
- Program the right extension time in your thermocycler based on sequence length, buffer, and enzyme
- 30 secs/kb for Q5 and 1 min/kb for OneTaq
Materials
- Thermocycler
- Q5 High-Fidelity 2X Master Mix
- Forward Primer (10 μM)
- Reverse Primer (10 μM)
- Template DNA
- MilliQ Water
Protocol
-
Set up the following reactions (for 25 μL reaction):
Reagents Volume (μL) Q5 High-Fidelity 2X Master Mix 12.5 Forward Primer (10 μM) 1.25 Reverse Primer (10 μM) 1.25 Template DNA Variable MilliQ Water Up to 25 μL Total: 25 μL -
Program the thermocycler using the following conditions:
Step Temperature (°C) Time (seconds) Initial Denaturation 98 30 25 cycles 98 5-10 50-72 10-30 72 30 seconds per 1kb Final Extension 72 2 minutes Hold 4 ∞ - Run a subsequent gel electrophoresis in order to confirm presence of the construct
Troubleshooting Tips
- Dilute one colony in 10 µL of DI water to not overload PCR with too much template DNA, otherwise the same as PCR
Materials
- NEB Monarch® DNA Gel Extraction Kit (NEB #T1120)
Protocol
Note: All centrifugation steps performed at 16000g.
- Excise the DNA fragment from the gel using a razor blade, weigh the sample, and transfer the sample to a 1.5mL microcentrifuge tube
- Add 4 volumes of Monarch Gel Dissolving Buffer to the tube with the gel (ie. 400μL for 100 mg agarose)
- If gel slice > 150mg, consider decreasing the Gel Dissolving Buffer to 3 or 3.5 volumes
- Incubate the sample between 37-55℃ (typically 50℃), inverting periodically until the gel slice is completely dissolved (often 5-10 minutes)
- Insert the column into a collection tube and load the sample onto the column, spin for 1 minute, then discard the flow-through
- Re-insert the column into the collection tube, add 200μL DNA Wash Buffer, and spin for 1 minute (discarding flow-through is optional)
- Repeat wash (Step 5)
- Transfer the column to a clean 1.5 microcentrifuge tube, make sure the tip of the column doesn't come into contact with the flow-through, but if in doubt, re-spin for 1 minute before placing the column into a clean microcentrifuge tube
- Add ≥ 6μL DNA Elution Buffer, let stand for 1 minute, and then spin for 1 minute to elute the DNA
Materials
- Strain of choice
- Growing medium of choice (LB+Amp, LB+Kan, or LB+Spec for E.coli; BG-11 or SOT for Cyanobacteria)
-
BG-11 media (recipe described below)
Stocks Per 500mL / 100mL / Liter NaNO3 75.0g (per 500mL) K2HPO4 4.0g (per 100mL) MgSO4·7H2O 7.5g (per 100mL) CaCl2·2H2O 3.6g (per 100mL) Citric Acid* 0.60g (per 100mL) Ammonium ferric citrate green* 0.60g (per 100mL) Na2EDTA 0.10g (per 100mL) Na2CO3 2.0g (per 100mL) Trace elements: H3BO3 2.86g (per Liter) MnCl2·4H2O 1.81g (per Liter) ZnSO4·7H2O 0.22g (per Liter) Na2MoO4·2H2O 0.39g (per Liter) CuSO4·5H2O 0.08g (per Liter) Co(NO3)2·6H2O 0.05g (per Liter) Medium Per Liter Stock solution 1 10.0mL Stock solutions 2-9 1.0mL each - Make up to 1 Liter with deionized water. Adjust pH to 9.0 with 1M NaOH or HCl. For agar, add 15.0g per liter of Bacteriological Agar. Autoclave at 15 psi for 15 minutes.
- Due to precipitation, larger volumes require stocks 5 & 6 to be autoclaved separately or filter sterilized and added to the sterile medium in the airflow cabinet.
- For nitrogen-fixing cyanobacteria, prepare standard BG-11 while omitting Stock 1
Protocol
- Add 35mL of BG-11 to a cell culture-treated flask
- Do a 3% passage: add ~1.0mL of cyanobacteria sample to the flask
- Incubate at 38℃ and 60 rpm, set the lights inside to a 12hr on/off cycle
- Check the OD’s every 12 hours (ie. 9am & 9pm)
- Depending on the OD, do 3-10% passages every few days
Troubleshooting Tips
- Let grow overnight
- For E.coli
- Take OD’s and mini prep after it reaches OD600 of >0.7
- When taking OD’s, fill cuvette to 850 µL
- For UTEX3154
- Switch to pasteur pipettes as opposed to micro pipettes to ensure sterility
- Increase the inoculant ratios
- Went from 3% inoculation to more than 5%
- When taking OD’s, fill cuvette to 600 µL to reduce volume lost in the culture
- We set the light at 333 µmol/s/m2
- Warm up BG-11 before making inoculants
- Optimal media is not always necessary
- BG-11 is not the optimal media, but they grew just fine
References
[1] A modular cloning system for standardized assembly of multigene constructs. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. PLOS ONE . 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765. PubMed PMID 21364738.
[2] Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Werner S, Engler C, Weber E, Gruetzner R, Marillonnet S. Bioeng Bugs. 2012 Jan 1;3(1):38-43. doi: 10.1371/journal.pone.0016765. PubMed PMID 22126803.
[3] “Zyppy Plasmid Miniprep Kit,” ZYMO RESEARCH, 2023. https://www.zymoresearch.com/products/zyppy-plasmid-miniprep-kit?srsltid=AfmBOopa0khOS21ey9b1XdMteeDLi8_AkEL4B1AiXEzgxBQaVL6doKVe (accessed Oct. 01, 2024).
[4] “Quick-DNA HMW MagBead Kit,” ZYMO RESEARCH, 2023. https://www.zymoresearch.com/products/quick-dna-hmw-magbead-kit?srsltid=AfmBOoo7CSVk-ji8O-e59cGYkvv6pm8aJsABiL7Xd419gHGh9WfpLeyo (accessed Oct. 01, 2024).
[5] “| UCSC - iGEM 2023,” Igem.wiki, 2023. https://2023.igem.wiki/ucsc/ (accessed Oct. 01, 2024).
[6] New England Biolabs, Neb.com, 2024. https://www.neb.com/en-us/protocols/2015/03/04/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1600 (accessed Oct. 01, 2024).
[7] New England Biolabs, Neb.com, 2024. https://www.neb.com/en-us/products/t1020-monarch-dna-gel-extraction-kit (accessed Oct. 01, 2024).
[8]CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax. Vasudevan R, Gale GAR, Schiavon AA, Puzorjov A, Malin J, Gillespie MD, Vavitsas K, Zulkower V, Wang B, Howe CJ, Lea-Smith DJ, McCormick AJ. Plant Physiology.2019 May;180(1):39-55. doi: 10.1104/pp.18.01401. PubMed PMID: 30819783.
[9] New England Biolabs, Neb.com, 2024. https://www.neb.com/en-us/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492 (accessed Oct. 01, 2024).
[10] New England Biolabs, Neb.com, 2024. https://www.neb.com/en-us/protocols/2012/09/06/protocol-for-onetaq-2x-master-mix-with-standard-buffer-m0482 (accessed Oct. 01, 2024).