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AMPLIFICATION OF GENOMIC DNA

Cell Lysis

We used Stratagene kit for lysing human cells and extracting DNA.

• Pellet up to a maximum of 1x 106 cells for 30 sec. in microfuge. If more than 1X 106 cells are used, the reagent volumes will have to be increased accordingly. For adherent cells, a trypsin treatment is generally used prior to this step to free the cells into suspension. Be careful to perform the trypsin step in a timely manner so that5 the cells do not remain in undiluted trypsin for a long period of time.

• Aspirate media and wash cells with 500 l of 1x PBS.

• Pellet cells 30 sec. in microfuge at 14, 000 rpm.

• Aspirate PBS and resuspend cells in 500 l of PBS. Repeat spin and aspiration steps.

• Resuspend cells in 100 l PBS and 200l sterile water.

• Lyse cells by heating to 950C for 10-15 minutes.

• Allow the cells to cool briefly by setting at room temperature for 5 min. And add 10 l of 10 mg/ml Proteinase K to each sample. Quick vortex to mix.

• Incubate at 550C for one hour.

• Inactivate Proteinase K by heating to 950c for 10 minutes.

• Spin down condensation.

• Store at -200c.

PCR Amplification

Biolabs Phusion High- Fidelity DNA polymerase was used for all PCR amplifications. The reactions and conditions are given below.

Conditions:

PCR amplification with linkers required an additional 50 M Mg2+ in the mastermix.

USER Amplification

Procedure

The USER mix components are mixed (Table in materials).The PCR product must be purified before used in USER cloning

● 2 µl of the USER mix is transferred to PCR tubes.

● The PCR products is added in equal amounts of each and incubated for 40 minutes at 37°C and for 30 min at 25°C.

Site-Directed Mutagenesis

• Purify template plasmid DNA from a dam+ Escherichia coli strain (to ensure that all GATC sites are methylated for later digestion with DpnI).

• Design forward and reverse primers that will bind to the region of DNA you want to mutate but that contain the modifications you wish to make. See the CAD tool PrimerX.

• Run a primer-extension reaction with a proof-reading, non-displacing polymerase such as Pfu DNA polymerase. This results in nicked circular strands of the plasmid.

• Cut up the template DNA with DpnI.

• Transform the circular nicked DNA into a highly competent strain such as XL1-Blue. These cells will repair the nicks and not restrict the unmodified product DNA.

• Select colonies with the correct DNA.

Purification of PCR Products

QIAquick PCR Purification Kit Protocol using a micro centrifuge was followed for purification of PCR products.

Procedure

• Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene.

• Place a QIAquick spin column in a provided 2 ml collection tube.

• To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.

• Discard flow-through. Place the QIAquick column back into the same tube. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.

• Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min.

• Residual ethanol from Buffer PE is completely removed by an additional centrifugation.

• Add 30 l of double distilled nuclease free water to elute the DNA and centrifuge again.

• Collect the flow through.

Analysis of DNA

Gel Electrophoresis

• Depending on the expected size of the fragments to be analysed, the percentage of gel is determined. For fragments more than 1.2 kb, a 1% gel and fragments less than 1.2kb 2% gel is used.

• To make a 1% gel, 0.7g of agarose is added to 70 ml of TAE buffer.

• The agarose is melted in a microwave until the solution becomes clear.

• The solution is cooled down until it can be held for 5 continuous seconds.

• 1 µl of ethidium bromide is added to the solution.

Electrophoresis Setting

• Ensure electrophoresis chamber is clean and dry, tape the sides (with Autoclave tape, NOT standard masking tape) to make watertight. Slot in the desired comb.

• Add gel solution to the chamber, and wait to set. The comb can then be removed from the chamber.

• Fill the electrophoresis apparatus half-full with 1x TAE buffer solution avoiding air bubbles.

• The gel is loaded with the samples, a negative sample, and the DNA ladder.

• Connect the electrodes to the apparatus. Set DC voltage at 100V and run for around 60 minutes (or until DNA separates sufficiently)

• View the gel in a gel exposer.

Gel Extraction

We used the QIAgen quick gel extraction kit; the protocol is adapted from the QIAquick Gel Extraction Kit Protocol.

Protocol

• The gel is exposed to blue-light to illuminate the DNA fragments (stained by ethidium bromide).

• The desired DNA band is identified and physically removed with a knife, cleaned with ethanol.( ensure to wipe the ethanol off the knife to avoid degradation of DNA)

• Add 3 volumes of buffer QG to 1 volume of gel. E.g.add 300 μl of Buffer QG to each slice of gel (volume of the slice is 100 μl, and weighs approximately 100 mg).

• Incubate at 52°C for 10 min (or until fully dissolved). To aid solvation, mix by vortexing the tube every 2–3 min during the incubation. When the gel is fully dissolved, ensure that the color of the mixture is yellow, same as that of the QG buffer.

• Add 1 gel volume of isopropanol to the sample and mix.

• Place a QIAquick spin column in a provided 2 ml collection tube.

• Apply each sample of DNA fragment to a QIAquick column, and centrifuge for 1 min at 13000 rpm.

• Discard flow-through and place QIAquick column back in the same collection tube.

• To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.

• Spin the column again to get rid of any residual ethanol.

• Add 30 ml of nuclease free water at the centre of the column and keep for 1 minute.

• Place the column in a collection tube and spin for 1 minute at 13000 rpm. Collect the flow through.