Team:UCL/Protocols

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Restriction digestion protocol

Calculate the amount of insert DNA required to maintain 1:3 backbone:insert molar ratio using formula below. For standard ligations use 50 ng of vector DNA, increase the amounts of DNA if unsuccessful.
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Prepare the ligation mixture:
Incubate at 16C for 30 minutes
Heat inactivate by incubating at 80C for 20 minutes
Keep on ice until ready to proceed with transformation protocol

(If using part from the distribution: resuspend the DNA in 10 ul of MiliQ water, making sure that it turns red. Wait 10 minutes before adding the DNA to cells)
Put a tube of NEB DH 5 alpha E. coli cells on ice and wait until they thaw completely. Divide the cells into 50 ul aliquotes.
Add 1 ul of plasmid DNA to 50 ul of cells.
Mix by carefully flicking the tube. Do not vortex or pipette in and out!
Place the mixture on ice for 30 minutes.
Heat shock the cells at 42 °C for 30 seconds and immediately put on back on ice.
Keep cells on ice for next 5 minutes. Do not mix.
Pipette 950 ul of SOC media kept at room temperature into the mixture. If SOC is not available, use LB.
Incubate the mixture at 37 °C for 60 minutes
Prepare plates with appropriate antibiotics. Bring plates to room temperature before plating. Use 2 plates per transformation reaction.
Plate 200 ul of cells on one plate.
Pellet the remaining cells and resuspend in 200ul of LB.
Plate the remaining cells on second plate.
Incubate plates overnight at 37 °C.

Measure 0.50 g of agarose
Measure 50 ml of 1x TAE buffer using measuring cylinder
Add agarose and TAE buffer to conical flask and gently mix
Microwave the flask for 1 min
Wait for the mixture to cool down slightly before proceeding
Add 10 ul of 10mg/ml ethidium bromide solution and mix
Assemble the casting tray and pour the gel into it
Wait around 30 minutes until gel gets solidified
Put the gel into the gel chamber and pour 1x TAE buffer until it is fully covered
Load 6 ul of DNA ladder to the first well.
Prepare the samples by adding appropriate volume of 6x gel loading dye and load them
Assemble the gel chamber and run the gel for 40 minutes at 120V
Visualise the gel using the gel visualizer

Digest at least 500 ng of each part according to the restriction digestion
Run the digested DNA on the gel according to gel electrophoresis protocol
Identify the parts that you want to ligate on a gel and cut the bands out using razor blade
Purify the excised bands using the commercial kit according to the manufacturer's instructions
Quantify the DNA yield using DNA nanodrop
Proceed to ligation

Prepare the PCR mix:
Set up the PCR cycles according to the following rules:
- Confirm the PCR by running 2 ul of the product on the gel according to the gel electrophoresis protocol