Team:University College London/LabBook/Week14

From 2012.igem.org

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==Tuesday 11.09.12==
 
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'''Aim:'''  To carry out a preparative digest of the insert and backbones in preparation of a 3A transformation using the 3A ligation products in order to be able to analyse whether ligation was successful. In addition,  carry out a 3A ligation using the digested inserts and backbones
 
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'''Method:'''
 
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==Wednesday 12.09.12==
==Wednesday 12.09.12==
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'''Method:'''
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'''Results:'''  
'''Results:'''  
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Revision as of 01:45, 27 September 2012

Contents

Sunday 09.09.12

Aim: Day 1 of Generating competent cells.


Method:


Day 1 of making competent cells

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)


Monday 10.09.12

Aim: Day 2 of Generating competent cells. The aim is to incubate cells in the presence of CaCl2 to prepare the cell wall, allowing it to become permeable to DNA for transformation.


Method:


Day 2 of making competent cells

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.

Tuesday 11.09.12

Aim: Day 3 of Generating competent cells


Method:

Day 3 of making competent cells

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

Wednesday 12.09.12

Aim: To carry out a transformation using the 3A ligation products in order to be able to analyse whether ligation was successful.


Method:


Transformation

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)

Thursday 13.09.12

Aim:


Picking of colonies from the ligation transformation plates


Methods:


Picking of colonies from plates

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.


Friday 14.09.12

Aim:


To check whether the 3A assembly ligation has been successful by purifying the overnight culture from the previous day, carrying out an analytical digest and analysing results on a gel.


Methods:


A mini-prep was carried out as per the following protocol:


MiniPrep of O/N culture


This was followed by a 10ul analytical digest as per the following:


Analytical Digest
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


Finally, results of the figest were run on a gel as follows:

Gel Electrophoresis

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


Results:


The following digram shows results of the analytical digest, with each purified DNA being cut once and twice in order to confirm identity:

BioBricks BioBrick Size (bp) Ligation size (bp) Plasmid Backbone Plasmid Expected Size (bp) Combined Size
BBa_K729001 & BBA_J23100 933 & 35 968 pSB1C3 2070 3038
BBa_K729001 & BBA_J23106 933 & 35 968 pSB1C3 2070 3038
BBa_K729001 & BBa_J23119 & BBa_B0034 933 & 35 & 12 980 pSB1C3 2070 3050
BBa_K729002 & BBa_J23119 & BBa_B0034 484 & 35 & 12 531 pSB1C3 2070 2601


Conclusion:


The bands on the gel indicate are not as expected. Hence, it is concluded that the ligation was unsuccessful.



Tuesday 11.09.12

Step 1 - Aim:


To generate enough plasmid backbone (pSB1C3) to be used in a 3A ligation. This is followed by a PCR clean-up.


Methods:


PCR of plasmid backbone

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



Step 2 - Aim:


To check whether PCR is successful. After the PCR reaction, we can detect the presence of the correct products by running a 1% gel electrophoresis.


Methods:


Gel Electrophoresis

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


Results:


The following gel results indicate whether PCR was successful.


UCL2012Week14B.png


Conclusion:

Since band sizes were as expected, the PCR was considered to be successful. Hence, the backbone was purified and used in the 3A ligation.


Thursday 13.09.12

Aim:

To generate enough linker (BBa_J23119 + BBa_B0034) to be used in a 3A ligation. This is followed by a PCR clean-up.


Methods:


PCR

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


'''Step 2 - Aim:''' To check whether PCR is successful. After the PCR reaction, we can detect the presence of the correct products by running a 1% gel electrophoresis. '''Methods:'''

Gel Electrophoresis

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

'''Results:''' The following gel results indicate whether PCR was successful. [[File:UCL2012Week14A.png]] '''Conclusion:''' Since we obtained the expected bands in the gel, the PCR was considered successful and the purified DNA used in the 3A ligation.

Friday 14.09.13

Aim:


To characterise nuclease activity BL21 cells


Method:

Nuclease characterisation
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

Results:


The following table shows readings of the diameters and OD at different time points:


Date Time Colony diameter/mm Halo diameter/mm Absorbance at 600 OD
Friday 12.30 0 0 0
Friday 16.30 0 0 0
Friday 19.30 0 0 0
Friday 22.30 0 0 0
Saturday 01.30 0.5 1.5 0.068
Saturday 04.30 1 2 0.098
Saturday 07.30 1.5 3 0.159
Saturday 10.30 1.5 3.5 0.192
Saturday 12.30pm 2 4 0.203
Saturday 14.30 2 4 0.209
Saturday 16.30 2.5 5 0.215


The average depth of the colonies was noted to be 0.5 - 1mm


Conclusion:


It can be seen that nuclease works as expected in BL21 cells. However, nuclease activity is less efficient when compared to nuclease activity in WNu cells. This is in line with expectations, since nuclease is found naturally in WNu cells but not in BL21 cells.