Site directed mutagenesis

Background: QuikChange site-directed mutagenesis from Agilent Technologies (California, USA) allows you to make single nucleotide changes.

Oligonucleotides are used that have an almost identical sequence to the region of interest, but also contain the relevant mutation(s). These bind to the plasmid of interest and a high fidelity DNA polymerase can then replicate both plasmid strands. However, the ends of the plasmid cannot be joined leaving it with staggered nicks. DpnI endonuclease is added to the samples as it targets specifically methylated and hemi-methylated DNA resulting in it degrading the parental strand (without the mutation). The DNA must then be transformed into bacteria in order to fix the nicks. It can then be harvested from these cells at a much higher quantity and ready for transfection.

Reagents:

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 QuikChange site-directed mutagenesis kit

 “Stellar” E. coli (Clontech, California, USA)

 SOC medium

 Oligonucleotide primers

 DNase-free water

 dNTPs, 2.5mM each

 Kanamycin (50mg/ml) containing agarose plates

Method: Created a 50µL reaction made up of 10x reaction buffer, 5-50ng plasmid, 125ng of forward and reverse primers, 0.5µL dNTP mix, 0.5µL

PfuTurbo DNA polymerase and water. This mix was then added to a PCR block and run using the following cycle:

1. Initial denaturation stage of 95ºC 30 seconds

2. 12 cycles of 95ºC for 30 seconds then 55ºC for 1 minute and finally 68ºC for 7 minutes (I minute per kb of plasmid length).

The samples were then transferred to ice for 2 minutes to cool the reaction.

0.5µL of 20,000 units/ml DpnI was then added to the reactants and gently mixed. The samples were then transferred to a water bath at 37ºC, for 1 hour.

During this I gently thawed “Stellar” E. coli on ice, 25µl was then transferred to two clean 1.5ml microcentrifuge tube. 1µl of water was added to 1 tube, and 1µl of treated DNA was added to the other. The tubes were then swirled to mix and left on ice for 30 minutes. The samples were then carefully placed in a water bath for 1 minute at 48ºC, then transferred to ice for 2 minutes. In sterile conditions 500µl of SOC medium was added to each tube and they were incubated at 37ºC for 1 hour with agitation at 225-250 rpm in an orbital incubator. The mixture was then spread on Kanamycin (50mg/ml) containing agarose plates and left for 16 hours at 37ºC. Any colonies could then be mini-prepped and sent for sequencing to check the mutation was as desired. More information on specific gene changes and primers used will be given in chapter 6.

53 2.1.20 Colony PCR

Background: Colony PCR is a simple and quick method of determining if an insert has successfully incorporated into a plasmid. Cells are lysed to release the DNA, allowing plasmids to serve as templates for an amplification reaction.

Primers can either flank the insert or copy the entire plasmid. The amplified DNA can then be run on a gel to determine if insert is present.

Reagents:

 Master mix per reaction: 0.3mM dNTPs, 0.5 µM of each forward and reverse primers, 1.5mM MgCl2, 0.25 units recombinant Taq polymerase (Thermo Fisher, Loughborough, UK) in the supplied buffer.

 DNase-free water.

Method: 10+ colonies were chosen from an antibiotic containing agar plate. The colonies were selected by pressing them with a 20µL pipette tip then moving that tip to an Eppendorf tube containing 100µL sterile water. The tube was then heated for 3 minutes at 98°C. 1µL of this DNA-containing water was then added to 9µL of master mix. The reactants were then transferred to a PCR block with cycling conditions:

1. Initial denaturation stage of 94ºC 2 minutes

2. 35 cycles of 94ºC for 45 seconds, 42ºC for 30 seconds and 72ºC for 90 seconds

3. Final elongation of 72ºC for 10 minutes

The products of this cycling were then run on a1% agarose gel with ethidium bromide. If the primers were flanking the gene then any products showed that the insert was present. If the primers replicated the full plasmid then the DNA ladder would show the molecular weight, which will be higher if the insert is present. If the fragment was as expected the sample was then mini-prepped and sequenced. More information on specific gene changes and primers used will be given in chapter 6.

54 2.1.21 In-fusion cloning

Background: In-Fusion Cloning Kits by Clontech (California, USA) are designed to allow fast cloning of a fragment, or fragments of DNA into a vector. Primers are designed that flank the gene of interest but leave overhangs of 15-bp that have known restriction enzyme sequences at the end. A restriction enzyme can then be used to cut your plasmid of interest leaving compatible ends of both your plasmid and gene fragment. The In-Fusion Enzyme then allows the DNA fragment to fuse into the plasmid efficiently and precisely by recognizing 15-bp overlaps at their ends. The resulting plasmid should be seamless with no unwanted base pairs so can be used immediately.

Reagents:

 In-Fusion cloning kit

 “Stellar” E. coli (Clontech, California, USA)

 Master mix per reaction: 0.2mM dNTPs, 0.5µM of forward and reverse primers and 0.5 units of Q5polymerase, in Q5 polymerase buffer

 SOC medium.

 PCR clean up kit.

 DNase-free water.

 Kanamycin (50mg/ml) containing agarose plates.

 Restriction enzymes and relevant buffers.

Method: Two mixes were made containing template DNA (1ng) and master mix, to a 25µL total volume. The reactants were then transferred to a PCR block with one of two cycling conditions 64ºC or 72ºC, as advised by the protocol:

64ºC cycle:

1. Initial denaturation stage of 98ºC 30 seconds

2. 30 cycles of 98ºC for 10 seconds, 56ºC for 15 seconds and 64ºC for 15 seconds

3. Final elongation of 64ºC for 2 minutes 72ºC cycle:

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1. Initial denaturation stage of 98ºC 30 seconds

2. 30 cycles of 98ºC for 10 seconds, 56ºC for 15 seconds and 72ºC for 15 seconds

3. Final elongation of 72ºC for 2 minutes

The plasmid was then double digested with two restriction enzymes. 1µg of the plasmid was incubated for 4 hours at 37°C with 20 units of each restriction enzyme in 50µL reactions with relevant buffer (New England Biolabs, Ipswich, USA). The products were subsequently purified using a PCR clean up kit, as per instructions. Both the products of the DNA fragments and cut plasmid were then run on a 1% agarose gel with ethidium bromide to verify both have worked.

I found the 72ºC cycle samples gave a stronger signal so continued with them samples and discarded the samples from the 64ºC cycle. 50ng of cut plasmid and 100ng of insert were then incubated with the relevant In-Fusion kit

component at 50°C for 15 minutes, in a 10µL reaction. During this Stellar” E.

coli were gently thawed on ice. After which, 25µl were transferred to two clean 1.5ml microcentrifuge tubes. In 1 tube 1µl of water was added and the other 1µl of treated DNA. The tubes were swirled to mix and then left on ice for 30

minutes. The samples were then carefully placed in a water bath for 1 minute at 48ºC then transferred to ice for 2 minutes. In sterile conditions 500µl of SOC medium was added to each tube and they were left at 37ºC for 1 hour with agitation at 225-250 rpm in an orbital incubator. The mixture was then spread on Kanamycin (50mg/ml) containing agarose plates and left for 16 hours at 37ºC. Any colonies could then be mini-prepped and sent for sequencing to check the mutation was as desired. More information on specific gene changes and primers used will be given in chapter 6.