Primer Design
General rules to follow in primer design: Aim for a Tm of ~60-64℃ This Tm is for the portion of the primer that anneals to the DNA itself. Any overhangs will increase the Tm, but don’t include it at first when calculating annealing temperature Try and keep the length of the primer that binds to the DNA insert anywhere between 18-30 base pairs in length. (Thermo Fisher 2019) Always check to ensure the annealing sequence of your designed primer doesn’t bind anywhere else on your sequence as this will result in the generation of two or more potential products.
Adding restriction sites to DNA fragments
Follow this informative guide from addgene if trying to add restriction sites to the ends of your DNA https://www.addgene.org/protocols/pcr-cloning/ Use this site to find the reverse complement https://www.bioinformatics.org/sms/rev_comp.html Examples of primers designed for Restriction digest are shown in the Notebook tab
Adding overhangs for Gibson Assembly
For Gibson assembly the benchling software (Benchlng.com) was used to generate assembled plasmids.
Follow this guide, provided by benchling, to generate Gibson Assembled Plasmids and export your primers https://help.benchling.com/en/articles/671242-clone-using-the-gibson-assembly-wizard
Centrifugation
When using any centrifuge, ensure to always balance both sides of the centrifuge (Protocol1)
Figure 1: Proper centrifugation alignment
Source: https://handling-solutions.eppendorf.com/sample-handling/centrifugation/safe-use-of-centrifuges/centrifuge-safety/ Never leave the centrifuge until it has gotten up to the speed it was set to If the centrifuge begins shaking or making rattling noises, stop the run immediately Never put any glass in the centrifuge and always ensure that all tubes placed inside are rated for the intended speed you are putting at Set your speed to xg instead of RPM as RPM values change with the size of the centrifuge
NanoDrop
All DNA concentrations were measured using a Fisher Scientific µDrop plate and compatible Nanodrop machine. The User protocol is outlined in the link below https://assets.fishersci.com/TFS-Assets/LPD/manuals/N12691_uDrop%20User%20Manual_Rev.4.0.pdf?_ga=2.88473944.2106651491.1634851105-976830180.1634851105
PCR
We followed the protocol from NEB for Taq DNA polymerase https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer#Product%20Information Use temperatures gradients for the annealing step to increase chances of success
Restriction Digestion
All restriction digestions were conducted using NEB cloner software https://nebcloner.neb.com/#!/ Choose the “Digestion” option Select the enzymes you plan to use in your digestion from the drop down menu Ensure the enzymes you are selecting match the ones you have in your lab Click on “Show detailed protocol” and you will be redirected to a page detailing the mastermix for a 50µL reaction Ex: (image 2)
Figure 2: Restriction Digest Mastermix Example
Make the mastermix in a PCR tube Cap the tube and put it in the thermocycler at the designated temperature Depending on what restriction enzymes you plan on using, incubation times will differ We recommend incubating overnight regardless of the incubation time listed This will ensure that the majority of your DNA is digested
Colony PCR
Follow same protocols as PCR, except switch out template DNA with bacterial colony Under flame, take a toothpick and lightly dab a colony Put the toothpick into the PCR master mix and swirl the toothpick to release the colony from the toothpick Continue as normal PCR
Electrophoresis 1% Agar
Electrophoresis 1% Agarose Add 60mL TAE Add 0.6g Agarose Heat in the microwave for 45 seconds. Until liquid is see through. Add next 6µL of SYBR safe stain Place a gel base between the solidifying stand and lock it into place. Make sure it is airtight. Pour in the TAE + Agarose + Safestain molten mix into the base. Place well mold and let dry for ~20 mins Once dry remove well mold To prepare DNA use the ratio of 1 part DNA 5 parts 5x loading dye if concentration is unknown If concentration is known add 100 ng of DNA in 25µLof ddH2O and 5µL loading dye Load 5µL of DNA ladder into the leftmost column and load 30µL of DNA dye mixture into subsequent wells. Run the gel at 100V, 240mAmps, and 30~40 mins
Gel band cutting and extraction
Gels were purified using the Qiagen QIAquick Gel Extraction Kit https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/dna-clean-up/qiaquick-gel-extraction-kit/
Ligation
Protocols were followed from NEB using T4 DNA ligase https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202
Transformation
In -80C freezer dh5 alpha comp cells. Get these and put on ice. Put in ice and thaw for 20 minutes Mix lightly tap the tube to mix after thawed Under sterile conditions add 1-5 µL of DNA to 50 µL of cells. (10 picograms to 100 nanograms of DNA) Incubate for another 20 minutes on ice. Heat shock for 45 seconds at 42 degrees. Set the water bath to this temperature. Under sterile conditions, add 250µL LB w/ no antibiotics Tape down in 37℃ shaker to rescue for 30 to 60 minutes. Immediately streak onto antibiotic plates that correspond to the plasmid.
Pouring Plates
Measure out 1L of ddH2O Add your LB + agar mix (40g per 1L of solution) Put in autoclave for 40min at 121℃ at 15psi Remove from the autoclave to cool. Once the bottle/liquid inside is cool to the touch, add your measured amount of antibiotics (From stock solution [100mg/mL] add 1mL for every 1L of LB+Agar solution). Carb plates are 1microL per 1 mL of LB. Then immediately start pouring before the gel starts to cool (Do this and last step under a flame). ~30mL per plate
Figure 3: Antibiotics and their Respective Concentrations
Streaking Plates
Under sterile conditions, would be best to do in the flow hood with the blower on, open the lid of the plate and aliquot 150 microL onto the side of the plate. Diagrams of streaking done below.
Figure 4: Our Method of Plate Streaking
Use the flat side of a 1000microL tip to spread out to cover half of the plate in step two of the diagram above. STEP 3.) This step will take some practice. Balance the tip with a light touch and do one solid line out of the smeared part to start the first streak. Streak back and forth until ⅓ of the other half of the plate is scratched. Do this very delicately, you do not want to gouge the agar at all. Do another single streak out of the first and start your second streak. You will need to rotate to the plate to make this more comfortable. Do these same steps for the third streak. Make sure most of the plate is covered by streaks or the ½ spread from diagram step 2.
Inoculation
Addgene protocol for inoculation was followed https://www.addgene.org/protocols/inoculate-bacterial-culture/
Gibson Assembly
Gibson assembly protocol from NEB was followed https://www.neb.com/protocols/2012/09/25/gibson-assembly-master-mix-assembly Transfection: Follow Thermofisher protocol: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/lipofectamine3000_protocol.pdf
References
Staff, Behind The Bench. “PCR Primer Design Tips.” Behind the Bench, 25 Sept. 2019, www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#:%7E:text=A%20go od%20length%20for%20PCR,or%20anneal%20to%20the%20target.