Difference between revisions of "Team:UCL/Protocols"

Agarose gel electrophoresis

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

Assembly of 2 parts using gel extraction

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

Polymerase Chain Reaction

1. 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
2. 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
3. Confirm the PCR by running 2 ul of the product on the gel according to the gel electrophoresis protocol

Gibson Assembly

1. 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
2. Convert the concentration of vector and inserts from ng/ul to pmol/ul using the following formula:
   
3. 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
4. Incubate the reaction at 50C for 15 minutes. Following incubation, put samples on ice.
5. Proceed to transformation protocol. Use 2 ul of the Gibson Assembly reaction mixture for transformation

Agar plate preparation

IPTG-induced protein expression

1. On the afternoon before the induction, start seed culture with appropriate antibiotic from glycerol stock and leave to incubate overnight at 37C shaking
2. 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
3. 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 )
4. When culture reaches OD of 0.4-0.6, add IPTG to a final concentration of 1 mM (=100ul of 100 mM stock)
5. Incubate induced culture at 30 °C for 4 hours
6. Split the culture into 4 falcon tubes (~25 ml each) and harvest the pellets by centrifugation for 20mins at max speed
7. Resuspend each pellet in 2 ml of lysis buffer, lyse by sonication (10 cycles of 10 sec with 10 sec breaks)
8. Centrifuge 20 min at max speed.
9. Transfer all supernatants to separate tube
10. Measure the concentration of protein in supernatant using Bradford Assay
11. Store in the freezer

Spectrophotometric assays

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