Team:University College London/LabBook/Week14

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 a falcons, with 5mls of Lysogeny Broth and 100ul 1M MgS04.

Step 2 – 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 3 - Cell culture: Culture your falcon tubes overnight at a temperature of 37 oC. Leave for no longer than 16 hours.

Step 1 – Innoculation: Inoculate 100mls of Lysogeny Broth in pre-warmed conical with 1ml of the overnight culture from Day 2

Step 2 – Incubation: Incubate for two hours in a 37oC shaker until the cells are at the early log phase of the growth curve. Measure absorbance on a spectrometer until A6000 is approximately 0.3.

Step 3 – Incubation: Transfer to a chilled sterile 50ml Falcon tube and incubate on ice for 10 minutes Step 4 – Centrifuge:

-Time: 5 mins -G – 3300 -Temperature: 180C

Step 5 – Incubate: Resus in 10ml of ice cold 0.1M CaCl2 in 15% glycerol and incubate on ice for 30 minutes.

Step 6 – Centrifuge: -Time: 5 mins -G – 3300 -Temperature: 180C

Step 7 – Transfer: Resus in 1ml of ice cold 0.1M CaCl2 in 15% glycerol. Transfer 100ul aliquots to pre-chilled, pre-labelled eppendorf tubes. Store at -70oC

Step 8 – Centrifuge -Time: 5 mins -G – 3300 -Temperature: 180C

Step 9 - Storage: Store cells at -80oC

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 - Picking Colony: Using a pipette select an isolated colony

Step 2 - Deposit: Dip the colony in 10ul of dH20, preferably in screw top container

Step 3 - Boiling Stage:Boil the dH20 100 for 5-10mins

Step 4 - Centrifuge:

Time: 1 min

RPM: 8000

Temp: 18oC

Step 5 - Remove Supernatant: The supernatant contains the DNA template.

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 - Thawing cells: Thaw all materials on ice

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

Insert Table

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: 1 hour

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

Temperature: 18oC

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 - Setting up PCR tubes: Thaw reagents and add to PCR tubes in the proportions described in the table below

PCR Components	Volume (ul)
5x Reaction Buffer	10
25mM MgCl2	4
10mM dNTPs	1
10uM Forward primer	5
10uM Reverse primer	5
DNA Polymerase	0.25
Nuclease Free Water	24.25
Template DNA	0.5
Total Volume	0.5

Step 2 - PCR program: Add PCR tubes to a thermocycler and run under the following conditions.

PCR conditions	Temp (oC)	Time (s)
Initial Denaturation (1 cycle)	95	30
Denaturation/Annealing/Extension (30 cycles)	95/55/72	10/25/120
Final Extension (1 cycle)	72	600
Hold	4	∞

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 - Setting up PCR tubes: Thaw reagents and add to PCR tubes in the proportions described in the table below

PCR Components	Volume (ul)
5x Reaction Buffer	10
25mM MgCl2	4
10mM dNTPs	1
10uM Forward primer	5
10uM Reverse primer	5
DNA Polymerase	0.25
Nuclease Free Water	24.25
Template DNA	0.5
Total Volume	0.5

Step 2 - PCR program: Add PCR tubes to a thermocycler and run under the following conditions.

PCR conditions	Temp (oC)	Time (s)
Initial Denaturation (1 cycle)	95	30
Denaturation/Annealing/Extension (30 cycles)	95/55/72	10/25/120
Final Extension (1 cycle)	72	600
Hold	4	∞

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

For BL21 cell line which has inserted nuclease the following steps were followed:

1. Prepare 11-16 plates (10ml LBAgar+10ul CMP )

2. Streak cells onto all plates at the same time

3. Incubate at 37°C

4. Apply hydrochloric acid (HCL) to the first plate before putting them 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. 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