Team:UCL/Protocols

Protocols

Restriction digestion protocol

Prepare restriction digestion mixture:
IDT gBlocks

- 10 ul of DNA (10 ng/ul)

- 2 ul of 10 x 2.1 buffer

- 0.3 ul of EcoR1

- 0.3 ul of Pst1

- 7.4 ul of milliQ H2O

Other digestions

- Required amount of DNA

- 1 ul of 10 x 2.1 buffer

- 0.3 ul of EcoR1

- 0.3 ul of Pst1

- add milliQ H2O up to 10 ul

(if using total volume greater than 10ul, increase the amount of buffer accordingly)

Incubate at 37C for an hour

Heat inactivate by incubating at 80C for 20 minutes

Run a sample of digested DNA on a gel in order to confirm digestion:
- 2 ul of DNA

- 1 ul of 6 x Gel Loading Dye

- 3 ul of milliQ H2O

If digestion is confirmed, proceed to ligation

Ligation 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:
- required amount of insert DNA

- required amount of vector DNA

- 1 ul of T4 ligase

- 2 ul of 10 x ligase buffer

- add milliQ H2O up to 20 ul

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

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.

Agarose gel electrophoresis

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

Assembly of 2 parts using gel extraction

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

Polymerase Chain Reaction

Prepare the PCR mix:
- 12.5 ul of 2 x Q5 PCR master mix

- 1.25 ul of 10 uM forward primer

- 1.25 ul of 10 uM reverse primer

- 2 ng of DNA to be PCRed

- add milliQ H2O up to 25

Set up the PCR cycles according to the following rules:
Initial denaturation

- 98C for 30 seconds

35 cycles

- 98C for 10 seconds

- 30 seconds at primer melting temperature

- 72C for 30sec/kb of PCRed fragment

Final extension

- 72C for 2 minutes

- Hold at 4C

Confirm the PCR by running 2 ul of the product on the gel according to the gel electrophoresis protocol

Gibson Assembly

When designing the gBlock fragments for Gibson Assembly, make sure that the fragments have ~20 bp overlap and that first and last insert fragment have ~20 bp overlap with respective ends of PSB1C3

Convert the concentration of vector and inserts from ng/ul to pmol/ul using the following formula:

Prepare the Gibson Assembly mixture:
- 0.08 pmol of each insert

- 0.04 pmol of vector

- 10 ul of Gibson Assembly mix

- add milliQ H2O up to 20 ul

Incubate the reaction at 50C for 15 minutes. Following incubation, put samples on ice.

Proceed to transformation protocol. Use 2 ul of the Gibson Assembly reaction mixture for transformation

Agar plate preparation

IPTG-induced protein expression

On the afternoon before the induction, start seed culture with appropriate antibiotic from glycerol stock and leave to incubate overnight at 37C shaking

The next morning, use 2 µl of seed culture to inoculate 100 ml of fresh media with appropriate antibiotic in shaker flask and grow until an OD600 nm of 0.4-0.6 is reached

If necessary, prepare these in the meantime :
- 50 ml stock of lysis buffer (25 mM TRIS-Cl, 2 mM EDTA, pH 7.6):
weight 0.151g of Tris base

add 45 ml of water

titrate with HCL to pH 7.6

fill up to 50 ml with water

add 29.2 mg of EDTA

- IPTG 100 mM stocks: (23.8 mg IPTG per 1ml ddH2O )

When culture reaches OD of 0.4-0.6, add IPTG to a final concentration of 1 mM (=100ul of 100 mM stock)

Incubate induced culture at 30 °C for 4 hours

Split the culture into 4 falcon tubes (~25 ml each) and harvest the pellets by centrifugation for 20mins at max speed

Resuspend each pellet in 2 ml of lysis buffer, lyse by sonication (10 cycles of 10 sec with 10 sec breaks)

Centrifuge 20 min at max speed.

Transfer all supernatants to separate tube

Measure the concentration of protein in supernatant using Bradford Assay

Store in the freezer

Spectrophotometric assays

- Assay for tryptophan hydroxylase (BBa_K1598002) activity: download protocol