Team:University College London/LabBook/Week15

Step 1 - Thawing cells: Thaw all materials on ice

Step 2 - Adding Ingredient: Add the following ingredients to autoclaved/sterile eppendorf tubes

Component	Amount (ul) (one enzyme used)	Amount (ul) (two enzymes used)
dH20	2.5	1.5
Buffer 1x	1	1
DNA template	5	5
BSA	0.5	0.5
Enzyme 1	1	2
Enzyme 2	N/A	1

Step 3 - Addition of BioBrick: Flick contents gently and centrifuge.

Step 4 - Centrifuge:

RPM: 14000

Time: 1 minute

Temperature: 18oC

Step 5 - Digest Program: Place the samples on a thermocycler under the following conditions:

RPM: 550

Time: 2.5 hours

Temperature: 37oC

Step 6 - Denaturing Enzymes: If you are not running the samples on a gel immediately, denature the restriction enzymes by running the samples on a thermocycler under the following conditions:

RPM: 550

Time: 25 minutes

Temperature: 65oC

Preparing the Gel

Step 1: Within a conical flask, add 3ml 50X TAE, 1.5g Agarose, and 150ml RO water

Step 2: Heat for 1 min in microwave. Swirl. Heat again for 30s. If solution is clear stop. Else repeat.

Step 3: Cool solution under running cold water.

Step 4: Add 20ul ethidium bromide (normal concentration of EB solution is 500ug/ul)

Step 5: Pour into a sealed casting tray in a slow steady stream, ensuring there are no bubbles

Running a gel

Step 6: Add 1 part loading buffer to five parts of loading sample

Step 7: Position the gel in the tank and add TAE buffer, enough to cover the gel by several mm

Step 8: Add 5ul of DNA ladder to lane 1

Step 9: Add samples to the remaining wells

Step 10: Run at 100 volts for 1hour and 15 minutes

Imaging the Gel

Step 11: Place gel in GelDoc 2000 chamber

Step 12: Turn GelDoc 2000 chamber on 

Step 13: From computer: Quantity One > Scanner > Gel_Doc_Xr>Manuqal Acquire

Step 14: Alter the exposure/settings to give a clear image.

TAE - Tris-acetate-EDTA

EDTA - ethylenediamine tetraacetic acid

Step 1 - Thawing cells: Thaw all materials on ice

Step 2 - Adding Ingredient: Add the following ingredients to autoclaved/sterile eppendorf tubes

Component	Amount (ul) (one enzyme used)	Amount (ul) (two enzymes used)
dH20	2.5	1.5
Buffer 1x	1	1
DNA template	5	5
BSA	0.5	0.5
Enzyme 1	1	2
Enzyme 2	N/A	1

Step 3 - Addition of BioBrick: Flick contents gently and centrifuge.

Step 4 - Centrifuge:

RPM: 14000

Time: 1 minute

Temperature: 18oC

Step 5 - Digest Program: Place the samples on a thermocycler under the following conditions:

RPM: 550

Time: 2.5 hours

Temperature: 37oC

Step 6 - Denaturing Enzymes: If you are not running the samples on a gel immediately, denature the restriction enzymes by running the samples on a thermocycler under the following conditions:

RPM: 550

Time: 25 minutes

Temperature: 65oC

Preparing the Gel

Step 1: Within a conical flask, add 3ml 50X TAE, 1.5g Agarose, and 150ml RO water

Step 2: Heat for 1 min in microwave. Swirl. Heat again for 30s. If solution is clear stop. Else repeat.

Step 3: Cool solution under running cold water.

Step 4: Add 20ul ethidium bromide (normal concentration of EB solution is 500ug/ul)

Step 5: Pour into a sealed casting tray in a slow steady stream, ensuring there are no bubbles

Running a gel

Step 6: Add 1 part loading buffer to five parts of loading sample

Step 7: Position the gel in the tank and add TAE buffer, enough to cover the gel by several mm

Step 8: Add 5ul of DNA ladder to lane 1

Step 9: Add samples to the remaining wells

Step 10: Run at 100 volts for 1hour and 15 minutes

Imaging the Gel

Step 11: Place gel in GelDoc 2000 chamber

Step 12: Turn GelDoc 2000 chamber on 

Step 13: From computer: Quantity One > Scanner > Gel_Doc_Xr>Manuqal Acquire

Step 14: Alter the exposure/settings to give a clear image.

TAE - Tris-acetate-EDTA

EDTA - ethylenediamine tetraacetic acid

Step 1 – Creating culture media: In a sterile environment, set up X numbers of falcons, each with 5mls of media.

Step 2 - Inoculating Colonies into a Selective Broth:: Add Yul of antibiotic to reach desired antibiotic concentration.

(For Ampicillin this is 50ug/ml, For Kanamycin it is 25ug/ml, for Tetracycline it is 15ug/ml, and for Chloramphenicol it is 25ug/ml)

Step 4 – Selecting a Colony: Select a clear, isolated colony and using an inoculation hoop scoop up a colony onto the tip. Deposit in the falcon tube

Step 5 - Culture: Culture your falcon tubes overnight at a temperature of 37 oC. Leave for no longer than 16 hours.

Step 1 - Pellet Cells: Pellet 1-5ml bacterial overnight culture by centrifugation at >8000rpm (68800 x g) for 3 min at room temperature (15-25oC)

Step 2 - Resuspend Cells: Resuspend pelleted bacterial cells in 250ul Buffer P1 and transfer to a microcentrifuge tube

Step 3 - Puncturing Cell Membrane: Add 250ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.

Step 4 - Neutralising buffer P2: Add 350ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.

Step 5 - Centrifuge:

RPM: 13000

Time:10 minutes

Temperature: 18oC

Step 6 - Centrifuge: Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60s and discard the flow-through.

Step 7 - Remove Endonucleases from Sample: Wash the QIAprep spin column by adding 500ul of Buffer PB. Centrifuge for 30-60s and discard flow-through.

Step 8 - Remove salts from sample: Wash the QIAprep spin column by adding 750ul of Buffer PE. Centrifuge for 3-60s and discard flow through.

Step 9 - Centrifuge:

RPM: 13000

Time:1 minute

Temperature: 18oC

Step 10 - Elute DNA: Place the QIAprep column in a clean 1.5ml microcentrifuge tube. To elute DNA, add 50ul Buffer EB to the centre of the spin column, let it stand for 1 min, and centrifuge for 1 min.

Step 1 - Addition of BioBrick: To the still frozen competent cells, add 1 - 5 µL of the resuspended DNA to the 2ml tube.

Step 4 - Incubation: Close tube and incubate the cells on ice for 45 minutes.

Step 5 - Heat Shock: Heat shock the cells by immersion in a pre-heated water bath at 37ºC for 10 minutes.

Step 6 - Incubation: Incubate the cells on ice for 2 minutes.

Step 7 - Add media: Add 1.5ml of Lysogeny Broth and transfer to a falcon tube.

Step 8 - Incubation: Incubate the cells at 37ºC for 1 hour at RPM 550. 

Step 9 - Transfer: transfer the solution back into a 1.5ml Eppendorf and centrifuge

RPM: 14000

Time: 2 minutes

Temperature (18-25oC)

Step 10 - Resuspend:Remove supernatant and resuspend in 100ul LB

Step 11 - Plating: Spread the resuspended cell solution onto a selective nutrient agar plate. Place the plates in a 37°C static incubator, leave overnight (alternatively a 30°C static incubator over the weekend)

Step 1 – Creating culture media: In a sterile environment, set up X numbers of falcons, each with 5mls of media.

Step 2 - Inoculating Colonies into a Selective Broth:: Add Yul of antibiotic to reach desired antibiotic concentration.

(For Ampicillin this is 50ug/ml, For Kanamycin it is 25ug/ml, for Tetracycline it is 15ug/ml, and for Chloramphenicol it is 25ug/ml)

Step 4 – Selecting a Colony: Select a clear, isolated colony and using an inoculation hoop scoop up a colony onto the tip. Deposit in the falcon tube

Step 5 - Culture: Culture your falcon tubes overnight at a temperature of 37 oC. Leave for no longer than 16 hours.

Step 1 - Pellet Cells: Pellet 1-5ml bacterial overnight culture by centrifugation at >8000rpm (68800 x g) for 3 min at room temperature (15-25oC)

Step 2 - Resuspend Cells: Resuspend pelleted bacterial cells in 250ul Buffer P1 and transfer to a microcentrifuge tube

Step 3 - Puncturing Cell Membrane: Add 250ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.

Step 4 - Neutralising buffer P2: Add 350ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.

Step 5 - Centrifuge:

RPM: 13000

Time:10 minutes

Temperature: 18oC

Step 6 - Centrifuge: Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60s and discard the flow-through.

Step 7 - Remove Endonucleases from Sample: Wash the QIAprep spin column by adding 500ul of Buffer PB. Centrifuge for 30-60s and discard flow-through.

Step 8 - Remove salts from sample: Wash the QIAprep spin column by adding 750ul of Buffer PE. Centrifuge for 3-60s and discard flow through.

Step 9 - Centrifuge:

RPM: 13000

Time:1 minute

Temperature: 18oC

Step 10 - Elute DNA: Place the QIAprep column in a clean 1.5ml microcentrifuge tube. To elute DNA, add 50ul Buffer EB to the centre of the spin column, let it stand for 1 min, and centrifuge for 1 min.

Software ND-1000 Model:

Step 1: Initialise the spectrophotometer by pipetting 1 µ of clean water onto lower optic surface, lowering the lever arm and selecting ‘initialise’ in the ND-1000 software

Step 2: Wipe and add elution buffer as negative control. Click blank in ND-1000 software

Step 3: Wipe and add 1 µl sample

Step 4: On the software set lambda to 260nm

Step 5: Lower the lever arm and click measure in ND-1000 software

Step 6: Take readings for concentration and purity

Step 7: Once measurement complete, wipe surface

Step 1 - Thawing cells: Thaw all materials on ice

Step 2 - Adding Ingredient: Add the following ingredients to autoclaved/sterile eppendorf tubes

Component	Amount (ul) (one enzyme used)	Amount (ul) (two enzymes used)
dH20	2.5	1.5
Buffer 1x	1	1
DNA template	5	5
BSA	0.5	0.5
Enzyme 1	1	2
Enzyme 2	N/A	1

Step 3 - Addition of BioBrick: Flick contents gently and centrifuge.

Step 4 - Centrifuge:

RPM: 14000

Time: 1 minute

Temperature: 18oC

Step 5 - Digest Program: Place the samples on a thermocycler under the following conditions:

RPM: 550

Time: 2.5 hours

Temperature: 37oC

Step 6 - Denaturing Enzymes: If you are not running the samples on a gel immediately, denature the restriction enzymes by running the samples on a thermocycler under the following conditions:

RPM: 550

Time: 25 minutes

Temperature: 65oC

For BL21 cell line that has been modified to contain nuclease:

1. Prepare 11-16 plates (DNAse - Agar + CMP)

2. Streak cells onto all plates at the same time

3. Incubate at 37°C

4. Apply HCl to the first plate before putting in the incubator (set time as zero)

5. Take a second reading after four hours, followed by six readings every 3 hours, and a final three readings every two hours.

6. When the reading is taken, observe the following: a. Diameter of the colony (once the diameter of the colony is measured, pick the colony and put it to grow in LB for nine hours) b. Diameter of the halo that is achieved once HCL is applied c. OD from a) d. Estimate of the depth of the colony on the agar plate

In order to characterise laccase, the following steps are carried out:

1) W3110 cells transformed with laccase and control W3110 cells are inoculated in 10mL of LB media, and incubated overnight at 37˚C in a 200rpm shaker

2) The O/N culture is then centrifuged at 6100g for 20 minutes, in order to extract the media containing laccase. The supernatant is retained.

3) In order to take readings, cuvettes are prepared with 2.2mL KH2PO4 buffer + 0.5mL Laccase supernatant + 0.3mL Syringaldazine, which is added immediately before readings are begun (as explained in step 4). A blank is created using 0.5mL of LB media instead of laccase supernatant.

4) The optical density at 530nm of each of the samples is measured at five minute intervals over 30 minutes.