Difference between revisions of "Team:Oxford/Protocols"

Protocols

Cloning

Antibiotics

Table 1: Antibiotic concentrations

• Weigh the solid antibiotic powder
• Add the appropriate amount of MilliQ and mix to dissolve
• Pour solution in a syringe with a 0.45μm syringe filter at the end of it and filter sterilise in plastic 1.5ml testubes
• Store in freezer (-20C)

Liquid Cultures of E. coli

Liquid cultures of E. coli are grown in LB,containing the appropriate antibiotics in a 37C, 225rpm incubator.

Agar Plates for E. coli

E. coli is grown on solid agar plates made from LB, and 2% agar containing the appropriate antibiotics.

Polymerase Chain Reaction (PCR)

PCR is used to amplify a specific region of DNA. A PCR reaction contains tem-plate genomic or plasmid DNA, a buffer containing MgCl2, dATP, dCTP, dGTP & dTTP (the nucleotides that are used to synthesize DNA, dNTP is used as a short-hand for all of the nucleotides), a DNA polymerase (an enzyme that can synthesis DNA using a single stranded DNA template) and a pair of primers. Primers are short pieces of DNA that mark the start and end of the region to be amplified, they bind to single stranded DNA and allow the DNA polymerase to bind and start synthesis.

Reaction mix:

This is an example, quantaties may vary depending on polymerase used, an . If DMSO is needed then the amount of MilliQ will need to be reduced by the amount of DMSO used (0 ml for 0%, 2.5 ml for 5%, 5 ml for 10%)

Table 2: PCR reaction mixture composition

Reaction protocol:

This is an example, times may vary depending on polymerase used.

Table 3: PCR reaction program. Annealing temperature dependent on Tm of primers

Notes:

• dNTP stock solution: 920 ml MilliQ, 20 ml each dATP, dCTP, dGTP, and dTTP.
• If the initial PCR conditions do not give sufficient PCR product then we have a gradient PCR machine which allows a range of annealing tempera-tures to be used for optimization.

DNA Gel Electrophoresis and Extraction

Agarose gel electrophoresis is used to separate DNA by size. Once the gel has been run we stain it with Ethidium bromide and then visualize the DNA using a UV light source DNA will show up as a bright white band. We use a razor blade to cut out any DNA bands which have the correct size and then purify them using the Qiagen gel extraction kit.

Pouring and running the gel:

• For 1% gels, add 2 g of agarose to 200 ml of ½ TBE buffer in a 500 ml duran bottle. Then heat the agarose in the microwave so that it dissolves. Use the “high” setting
• Once dissolved, transfer the bottle to the 55 C water bath to cool (~30 mins)
• Whist the gel is cooling prepare the gel tray
• Pour the gel, you’ll need 100 ml for a small gel, and 200 ml for a large gel
• Leave to set for ~30 mins
• Place the gel in a gel tank, and add ½ TBE buffer to cover
• Add 5X DNA loading dye to samples – e.g. add 10 ml to a 50 ml PCR
• Load DNA ladder and samples into the wells. Each large well will hold 30 ml so you’ll need 2 wells per 50 ml PCR
• Run the gel. Don’t forget black electrode (-ve) at back, and red (+ve) at the front. Use 100 - 140V
• Put the gel into the Ethidium bromide to stain for 10-20 mins

Visualising DNA on the gel

Having stained the gel with Ethidium bromide, wash the gel in water. Use the GelDoc to photograph the gel (photos of gels should kept in you lab book).

• Put gel in the machine
• Choose the “agarose gel” button and then the size of the gel
• Wait for the machine to process your choices
• Edit, save and/or print an image of the gel

To cut bands out of the gel use the UV transilluminator (MAKE SURE YOU ARE WEARING A UV FACE SHIELD).

• Close lights
• Use a razor to cut the target band

DNA extraction from gels

We used a slightly modified version of the the QIAGEN gel extraction kit (catalogue no. 28704 or 28706) protocol provided with the kit.

• Notes before starting the protocol
• Add ethanol (96-100%) to the PE buffer before its use
• All centrifugation steps are carried out at 13000 rpm

• Excise the target DNA bands from the gel using a razor
• Weight the gel slice in a plastic testube. Add 3 volumes of Buffer QG to 1 volume of gel (100mg gel ≈ 100μl)
• Incubate at 50C for 10min at 350-400rpm until the gel is completely dissolved. Every 2 minutes check the gel and vortex to speed up the dissolution. After the gel is dissolved the mixture should be yellow. If it is orange or violet, add 10μl of 3M sodium acetate, pH 5.0 and mix. The solution should now be yellow
• Add 1 volume isopropanol to the sample and mix/vortex
• Apply the DNA from the testube in a QIAquick column which is in a QIAquick spin tube
• Centrifuge for 1 minute and discard flow-through
• Add 500μl Buffer QG to the column and centrifuge for 1 min. Discard flow through
• Add 750μl of Buffer PE (note: with ethanol dissolved in it) in the column and centrifuge for 1 minute
• Take column out of the centrifuge and let it on the bench for 2-5 minutes so that any residual ethanol has evaporated
• Spin again for 1 minute to make sure that all ethanol has been removed
• Place the QIAquick column in a 1.5ml plastic testube
• To elute DNA, add 30μl of MilliQ to the QIAquick column. Let it settle for 1 minute so that DNA has dissolved
• Centrifuge for 1 minute
• Pipette the flowthrough from the bottom of the testube to the QIAquick column and centrifuge for a second time to make sure you have maximal DNA dissolution
• DNA is ready of storage or further experimentation

Restriction Enzyme Digests

All the restriction enzymes we use are from New England Biolabs (NEB), please check either one of their catalogues or their website to find out which buffer is optimal for the enzyme being used. If using two REs together check to make sure the buffer used is compatible with both enzymes (can use the NEB website to find out which buffer is best: https://www.neb.com/tools-and-resources/ interactive-tools/double-digest-finder.

Restriction enzyme digest reaction mixtures are described in tables 4 and 5. Incu-bate at 37 C for at least 2 hours or overnight.

Table 4: Restriction enzyme digest reaction mixture composition

Table 5: Restriction enzyme digest reaction mixture for confirmation of ligation

Run digested plasmid on a gel, take photo, and gel extract.For digested PCRs just gel extract (no need to run on gel).

Ligation

An example DNA ligation mixture is described in table 6, the volume of plasmid and insert DNA was adjusted to give a specific molar ratio of insert to plasmid, usually 3:1. Use the nano-drop to determine the concentration of the plasmid and insert DNA.

Table 6: DNA ligation reaction mixture

Reaction mixtures should be incubated at 16 C overnight.

E. coli Competent Cell Preparation

Day 1

• Inoculate 5 ml LB in test tube with the E. coli strain to be made competent. Add the appropriate antibiotic to the solution to select for the bacteria that have the target construct
• Grow overnight in a 37C, 225 rpm shaker

Day 2

• Inoculate 100 ml of LB using 1 ml of the overnight culture. Incubate at 37C
• Pre-chill eppendorfs. 20 needed per 100ml
• Grow culture to OD600nm0.4-0.6.. This takes ~ 2-2.5 hours
• Decant culture into 2 50 ml falcon tubes
• Spin at 2000 rpm for 20 mins (4 C)
• Discard supernatant
• Gently resuspend pellets in 1 ml TFB I. (Both TFB I and II should be on ice!)
• Add a further 16 ml TFB I to each falcon
• Leave on ice for 20 mins
• Spin for 10 mins at 2000 rpm (4C)
• Discard supernatant
• Gently resuspend pellets in 2 ml TFB II
• Aliquot 200 ml into each pre-chilled eppendorf
• Flash freeze
• Store at -80 C

Transforming E. coli with Plasmid DNA

Plasmids are introduced into the E. coli through a cold/heat shock process. The E. coli cells are then grown in LB growth medium for one hour to allow cells with a plasmid to start making a protein that makes the cells antibiotic resistant. The cells are then spread on solid growth medium (LB-Agar) containing antibiotic and grown overnight at 37 C. Only cells with a plasmid will form colonies.

Different strains of E. coli are used for different purposes e.g. XL1 Blue or DH5a for making stocks of plasmid DNA or BL21 cells for protein expression.

If transforming a ligation add no more than 25 ml of ligation to 100 ml of cells. If transforming a Gibson product add 5 ml to 50 ml of cells.

• Thaw aliquots of competent E. coli cells on ice. Need 100 ml per transformation (one 200 ml aliquot = 2 tranformations).
• Thaw plasmid DNA
• Thaw appropriate antibiotic
• Once cells are thawed split into 100 ml
• Add 1 ml of plasmid DNA
• Incubate on ice for 30 mins. (Now is a good time to melt the LB agar to make the plates you’ll need. Two plates per transformation.)
• Heat shock (45 s in 42 C water bath, then on ice for 1 min.)
• Add 800 ml LB and incubate at 37 C for 1 hour
• Spread 100 ml of the cells onto a plate using sterile glass beads, then spin down the remaining and resuspend in 100 ml . Spread this 100 ml onto a second plate. This should be done in a sterile environment so work near a flame or use the flow cupboard
• Incubate plates at 37C overnight

Purifying Plasmid DNA from E. coli

Day 1

• Pick single colonies from a plate into 5 ml LB with appropriate antibiotic
• Grow at 37 C overnight ina 37C 225rpm incubator

Day 2

We used the QIAGEN Miniprep kit protocol (catalogue no. 27104 or 27106) provided in the kit.

• Notes before starting the protocol
• Add LyseBlue (Dye) to buffer P1 at a 1:1000 ratio
• Add the RNase A provided in the kit to buffer P1 and mix. Store at 2-8C after use
• Add ethanol (96%-100%) to buffer PE before use
• All centrifugation steps were revolutions are not mentioned are carried out at 13000 rpm

• Pour 1.5ml of the overnight bacterial solution in a plastic testube. Centrifuge and discard supernatant. Repeat to get a larger pellet and therefore extract more DNA
• Resuspend pellet in 250μl of Buffer P1 and transfer the solution to a microcentrifuge tube
• Add 250μl of P2 buffer and mix by inverting the tube 4-6 times until the solution becomes blue. Proceed to next step in less than 5 minutes
• Add 350μl buffer N3 and mix immediately by inverting the tube again. The solution should turn colourless
• Centrifuge for 10 minutes
• Apply 800μl of the supernatant in a QIAprep 2.0 spin column by pipetting. Centrifuge for 1 minute and discard the flowthrough
• Add 500μl of buffer PB. Spin for 1 minute and discard flow through
• Wash the QIAprep 2.0 spin column by adding 750μl of buffer PE (with ethanol in it). Centrifuge for 1 minute and discard flowthrough
• Leave on bench for 2-5 minutes so residual ethanol can evaporate. To ensure no residual ethanol is left centrifuge for a minute again and discard any possible flowthrough
• Transfer the spin column in a fresh 1.5ml testube
• Add 30μl of dH2O and leave it for a minute so that DNA can dissolve. Centrifuge for 1 minute. Pipette the flowthrough back into the spin column for a second time to maximize DNA dissolution
• Use nano-drop to check DNA concentration if needed. Blank with MilliQ first and then add 2μl of the DNA to be measured
• Store at -20 C

Testing