Team:Kent/Experiments

Experiments and Protocols

Competent Cells

Overview

Competent cells are ready to use bacterial cells that possess altered cell walls by which foreign DNA can be passed through easily. E. coli cells that have been specially treated to transform efficiently.

Materials

3ml 1M MnCl₂

15ml 1M CaCl₂

60ml 50mM MES

45ml glycerol

177ml ddH2O

Procedure

1. Back-dilute overnight culture of VS45 cells to OD600 0.1 in 250 ml LB broth
2. Grow cells at 37˚C to OD600 0.6 and then harvest by centrifugation.
3. Resuspend the cells in 100 ml of prechilled buffer and incubate on ice for 60 minutes.
4. Harvest again by centrifugation (at 4˚C), and resuspend in 5 ml of pre-chilled buffer.
5. The resuspended cells can then be aliquoted (on ice), frozen using dry ice or liquid nitrogen, and stored at -80˚C.

Transformation Protocol

Overview

Transformation is the process by which a foreign DNA is introduced into a cell.

Materials

Resuspended DNA

Competent cells

2ml tube

42˚C water bath

Petri dishes with LB agar and appropriate antibiotic

37˚C incubator

10pg/ul RFP Control

Procedure

1. Thaw the competent cells on ice
2. Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
3. Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently.(Make sure to keep the competent cells on ice. )
4. Add 1 µL of the RFP Control to your control transformation.
5. Close tubes and incubate the cells on ice for 30 minutes.
6. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
7. Incubate the cells on ice for 5 minutes.
8. Add 200 μl of SOC media (making sure that the broth does not contain antibiotics and is not contaminated) to each transformation
9. Incubate the cells at 37˚C for 2 hours while the tubes are rotating or shaking.2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.
10. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
11. For the control, label two petri dishes with LB agar (AMP). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
12. Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up. (Incubating for too long starts to break down the antibiotics and un-transformed cells will begin to grow.)
13. Pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.
14. Count the colonies on the 20 μl control plate.

Plasmid Miniprep

Overview

The Miniprep is for purification of molecular biology grade plasmid DNA, this provides a rapid method to purify plasmid DNA using silica membrane column.

Materials

Qiagen Miniprep Kit

Procedure

Procedure was carried out according to the manufacturer’s instructions, summarized below:

Add the provided RNase A solution to Buffer P1.

Mix the solution and store at 2–8°C

Add ethanol (96–100%) to Buffer PE before use

1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer it to a microcentrifuge tube.
3. Add 250 μl 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. If using LyseBlue reagent, the solution will turn blue.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30–60 s and discard the flow-through.
7. Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through.
8. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through
9. Centrifuge for 1 min to remove residual wash buffer.
10. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.
11. Add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

PCR

Overview

PCR is a method to amplify sections of DNA fragments

Materials

50µl PCR reaction

300nM of forward and reverse primers

25µl 2x Master Mix

21µl sterile MQ H₂O

Procedure

Pick a colony and resuspend in 50µl of MQ H₂O

Take 1µl of the cell suspension and add to 50µl PCR reaction

PCR cycles:

1. 95º for 5 minutes

2. 95º 19 seconds

3. 50º for 30 seconds

4. 68º for 1.5 minutes

5. 68º for 5 minutes

steps 2-4 last for 35 cycles

PCR Purification

Overview

This method is for rapid and efficient purification of DNA from the PCR reaction to mainly remove salts and enzymes from the PCR product.

Materials

Thermoscientific PCR Purification Kit

Procedure

Procedure was carried out according to the manufacturer’s instructions, summarized below:

1. Add 1:1 volume of binding buffer to the PCR mixture and mix thoroughly.

2. Transfer around 800 μl of the sample mixture to GeneJET purification column. Centrifuge for 1 minute and discard the flow-through.

3. Add 700 μl of wash buffer to the GeneJET purification column. Centrifuge for 1 minute and discard the flow-through.

4. Centrifuge for another 1 minute to remove the residual wash buffer completely.

5. Place the GeneJET purification column in a clean 1.5 mL microcentrifuge tube. Add 50 μl of elution buffer to the centre of the column to elute the DNA. Centrifuge for 1 minute.

6. Discard the column and store the purified PCR DNA at -20°c.

Ligation

Overview

A method of joining two DNA strands

Materials

DNA strands

DNA dilution buffer

T4 DNA Ligation buffer

T4 DNA Ligase

Procedure

Dissolve the DNA in 1x concentrated DNA dilution buffer to a total volume of 10µl

Add 10µl of T4 DNA Ligation buffer and mix thoroughly

Add 1µl of T4 DNA Ligase and mix thoroughly

Incubate at 15-25ºC for 15 minutes

AFM Imaging

Overview

A method of viewing the topography of a sample using Atomic Force Microscopy, generating a 3-D image of our nano-wire products.

Materials

VS45 E. coil cells transformed with gene encoding our fusion protein in pVS72 plasmid backbone.

Chloramphenicol and Amp combined plates (non-inducing)

Chloramphenicol and Amp combined plated, with 20% w/v Arabinose and 1mM IPTG (inducing)

1x PBS

0.5ml water

AFM Preparation procedure

Grow VS45 cells containing PVS72 on an imaging plate (see below) and a non-inducing plate for 5 days at 22ºC

Pipet 25µl 1x PBS onto a spot of bacterial cells

Pipet the PBS up and down 5 times

Transfer 20µl of each resuspension into an eppendorf tube

Place 10µl of each sample onto a freshly cleaved mica

Incubate for 40 minutes

Wash with 0.5ml of water

Gibson Assembly

Overview

Gibson assembly is a method of joining DNA fragments in a single reaction.

Materials

2-10µl of synthesised DNA + linearized vector (25-100ng of vector and at least 2-fold excess inserts)

10µl of Gibson Assembly Master Mix (NEB)

Deionised H₂O

Assembly Procedure

Add 2-10µl of PCR Fragments + linearized vector to 10µl of Gibson Assembly Master Mix

Add Deionised H₂O as necessary to bring the total reaction volume to 20µl

Incubate the samples at 50ºC in a thermocycler for 15 minutes

Store the samples at -20ºC

Transform the product into competent cells using the Gibson Assembly Transformation Protocol

Transformation protocol below:

Thaw competent cells on ice

To the competent cells, add 2µl of the chilled assembly product

Mix the Assembly product with the competent cells by pipetting up and down 4-5 times

Incubate the mixture on ice for 30 minutes

Heat shock at 42ºC for 30 seconds

Return the tubes to ice for 2 minutes

Add 950µl of SOC media to the tube

Incubate at 37ºC for 60 minutes, whilst shaking

Warm appropriate selection plates to 37^oC

Spread 100µl of cells onto the plates

Incubate overnight at 37ºC

Agar Plates

Overview

The method to produce Agar plates, used to grow our bacteria on.

Materials

For a 1L mixture:

15g Agar

10g Sodium chloride

10g Tryptone

5g yeast

1L water

Optional Materials For Specialized Plates

Antibiotic plates:

Ampicillin (Amp) – 100mg/mL

Chloramphenicol (Chl) – 25mg/mL

Plates for imaging of amyloid fibrils (imaging plates):

Amp – 100mg/mL

Chl – 25mg/mL

L-Arabinose - 20% w/v

IPTG – 1mM

Congo red plates:

Same as imaging plates with the addition of: 10µg/mL Congo red

Haem soft plates for conductivity testing (Haem plates):

Amp – 100mg/mL

Chl – 25mg/mL

L-Arabinose - 20% w/v

IPTG – 1mM

Hemin – 0.6µg/mL

Procedure

Pour water and components into a 1L Duran flask and mix thoroughly

Autoclave at 126°C and allow to cool to around 50°C before pouring into sterilised agar plates. If making specialised plates, add the extra ingredients now.

Cells grown in LB broth overnight – 1mL for every 50mL of agar

DNA Restriction Digestion

Overview

It involves cutting the DNA with restriction enzymes to use the fragments for other processes such as ligation and Gibson assembly. We carried this preparative digest as well as diagnostic digest to confirm the sizes our DNA fragments.

Materials

10× concentrate NEB buffer 2

Restriction enzymes EcorI (concentration 10 U/μl and PstI (concentration 10 U/μl)

Sterile MQ water

Procedure

1. Take 5 μl of the sample (plasmid or fragment)

2. Add 1 μl of Ecor1 and Pst1 each.

3. Add 1 μl of 10× buffer 2 and 2 μl of water to make up the volume of the digest to 10 μl.

4. Incubate the digest at 37°c for 2 hours.

5. Optional step: If the digest sample is required for ligation reaction, heat inactivate the restriction enzyme by incubating at 80°c for 20 minutes.

Agarose Gel

Overview

A gel used to separate DNA fragments based on size

Materials

0.6g agarose

7.5μl Sybr safe

75ml TBE

Procedure

Add 0.6g agarose to 75ml TBE in a flat-bottomed flask and swirl to mix.

Microwave for 1 minute and see if it goes clear.

Microwave for a few more seconds if not yet clear.

Let the solution cool to 50°C and add the Sybr safe.

Set up the gel tank, adding the two spacers to the ends of the gel.

Then use a Pasteur pipette to line the edges of the gel tank and wait for it to set.

Once it is set, pour the rest of the liquid into the tank, between the spacers.

Add the comb at one end.

Once it is set, slowly pull out the comb and spacers.

Add cold TBE to the gel, covering it by 2-5ml.

Fill the wells with sample mixed with sample buffer in the proportions indicated for the sample buffer used.

Gel Extraction

Overview

This procedure is for rapid and efficient purification of the DNA fragments, cut and isolated from the agarose gel.

Materials

Binding Buffer

Wash buffer (diluted with ethanol)Elution buffer

GeneJET purification columns

Procedure

1. Excise DNA fragment using a clean scalpel from the agarose gel. Ensure to cut close to DNA to minimize gel volume.

2. Weigh the excised gel and add 1:1 volume of binding buffer to gel slice.

3. Incubate the gel mixture at 50°c to 60°c for 10 minutes to dissolve the gel completely.

4. Transfer up to 800 μL of solubilized gel solution to GeneJET purification column. Centrifuge for 1 minute and discard the flow-through.

5. Add 700 μl of wash buffer to the GeneJET purification column. Centrifuge for 1 minute and discard the flow-through.

6. Centrifuge for 1 minute to remove residual wash buffer completely.

7. Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge tube. Add 50 μl of elution buffer to the center of the column. Centrifuge for 1 minute.

8. Discard the GeneJET purification and store the purified DNA at -20°c.