Team:University College London/LabBook/Week11

For WNu cell line which has native secreted nuclease activity:

1. Prepare 11-16 plates (10ml DNAse - Agar + 10ul AMP + 10ul 1M IPTG). IPTG induces the lac promoter which in turn activates the transcription of nuclease.

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 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

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

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

== Phusion High Fidelity Polymerase ==

Component	20 µl Reaction	50 µl Reaction	Final Concentration
Nuclease-free water	to 20 µl	to 50 µl
5X Phusion HF or GC Buffer	4 µl	10 µl	1X
10 mM dNTPs	0.4 µl	1 µl	200 µM
10 µM Forward Primer	1 µl	2.5 µl	0.5 µM
10 µM Reverse Primer	1 µl	2.5 µl	0.5 µM
Template DNA	variable	variable	< 250 ng
DMSO (optional)	(0.6 µl)	(1.5 µl)	3%
Phusion DNA Polymerase	0.2 µl	0.5 µl	1.0 units/50 µl PCR

Thermocycling conditions for a routine PCR: Initial denaturation: 98°C 30 seconds

25–35 cycles: 98°C 5–10 seconds 45–72°C 10–30 seconds 72°C 15–30 seconds/kb

Final extension: 72°C 5–10 minutes

Hold: 4°C

The first digest was carried out in order to prepare the Upstream Part for ligation through an EcoRI-HF™ and SpeI preparative digest:

Ingredient	Amount
Upstream Part Plasmid	500 ng
EcoRI-HF	1 µl
Spel	1 µl
10X NEBuffer 2	5 µl
100X BSA	0.5 µl
H2O	to 50 µl

Another digest was carried out in order to prepare the downstream DNA for ligation through an XbaI/PstI preparative digest:

Ingredient	Amount
Downstream Part Plasmid	500 ng
Xbal	1 µl
Pstl	1 µl
10X NEBuffer 2	5 µl
100X BSA	0.5 µl
H2O	to 50 µl

Another digest was carried out to prepare the backbone plasmid through an EcoRI-HF/PstI preparative digest:

Ingredient	Amount
Destination Plasmid	500 ng
EcoRI-HF	1 µl
PstI	1 µl
10X NEBuffer	2.5 µl
100X BSA	0.5 µl
H2O	to 50 µl

For each of the digests above, incubate all three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.

Ligate the Upstream and Downstream Parts into the digested Destination Plasmid, using the following:

Ingredient	Amount
Upstream Part digestion	2 µl
Destination Plasmid digestion	2 µl
10X T4 DNA Ligase Buffer	2 µl
T4 DNA Ligase	2 µl
H2O	11 µl

After this is prepared, incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes.

1. Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert.)

COMPONENT	20 μl REACTION)
10X T4 DNA Ligase Buffer	2 μl
Vector DNA (3 kb)	50 ng (0.025 pmol)
Insert DNA (1 kb	50 ng (0.076 pmol)
Nuclease-free water	to 20 μl
T4 DNA Ligase	1 μl

2. The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

3. Gently mix the reaction by pipetting up and down and microfuge briefly.

4. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

5. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).

6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.