Team:University College London/LabBook/Week9

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 1.5-5ml bacterial culture containing the plasmid by centrifugation g = 6000

Time = 2 min

Temperature = (15-25oC)

Step 2 - Resuspend Cells: Add 250ul S1 to the pellet and resuspend the cells completely by vortexing or pipetting. Transfer the suspension to a clean 1.5ml microcentrifuge tube.

Step 3 - Puncturing Cell Membrane: Add 250ul S2 gently mix by inverting the tube 4-6 times to obtain a clear lysate. Incubate on ice or at room temperature for NOT longer than 5 min.

Step 4 - Neutralising S2: Add 400ul Buffer NB and gently mix by inverting the tube 6-10 times, until a white precipitate forms.

Step 5 - Centrifuge:

g: 14000

Time:10 minutes

Temperature: (15-25oC)

Step 6 - Centrifuge: Transfer the supernatant into a column assembled in a clean collection tube (provided. Centrifuge:

g = 10,000

Time: 1 minute

Temperature: (15-25oC)

Step 7 - Wash Column: Discard the flow-through and wash the spin column by adding 700ul of Wash Buffer. Centrifuge:

g - 10,000

Time - 1 minute

Temperature: (15-25oC)

Step 8 - Remove residual ethanol: Centrifuge:

g - 10,000

Time - 1 minute

Temperature: (15-25oC)

Step 9 - Elute DNA: Place the column into a clean microcentrifuge tube. Add 50-100ul of Elution Buffer, TE buffer or sterile water directly onto column membrane and stand for 1 min. Centrifuge:

g - 10,000

Time - 1 minute

Temperature: (15-25oC)

Step 10 - Storage: Store DNA at 4oC or -20oC

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 – 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 1.5-5ml bacterial culture containing the plasmid by centrifugation g = 6000

Time = 2 min

Temperature = (15-25oC)

Step 2 - Resuspend Cells: Add 250ul S1 to the pellet and resuspend the cells completely by vortexing or pipetting. Transfer the suspension to a clean 1.5ml microcentrifuge tube.

Step 3 - Puncturing Cell Membrane: Add 250ul S2 gently mix by inverting the tube 4-6 times to obtain a clear lysate. Incubate on ice or at room temperature for NOT longer than 5 min.

Step 4 - Neutralising S2: Add 400ul Buffer NB and gently mix by inverting the tube 6-10 times, until a white precipitate forms.

Step 5 - Centrifuge:

g: 14000

Time:10 minutes

Temperature: (15-25oC)

Step 6 - Centrifuge: Transfer the supernatant into a column assembled in a clean collection tube (provided. Centrifuge:

g = 10,000

Time: 1 minute

Temperature: (15-25oC)

Step 7 - Wash Column: Discard the flow-through and wash the spin column by adding 700ul of Wash Buffer. Centrifuge:

g - 10,000

Time - 1 minute

Temperature: (15-25oC)

Step 8 - Remove residual ethanol: Centrifuge:

g - 10,000

Time - 1 minute

Temperature: (15-25oC)

Step 9 - Elute DNA: Place the column into a clean microcentrifuge tube. Add 50-100ul of Elution Buffer, TE buffer or sterile water directly onto column membrane and stand for 1 min. Centrifuge:

g - 10,000

Time - 1 minute

Temperature: (15-25oC)

Step 10 - Storage: Store DNA at 4oC or -20oC

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

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

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

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

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