Remember timings! All things must be requested before 4pm the day before they are needed
Stocks/kits/plates
require access to ULT
material to autoclave needs to be ready before 12
Plasmids:
pUC19 needed for cloning AND as negative control in all transformation steps
pSDC115 needed as positive control
E.coli strains:
DS941 regular cloning (LB)
DS945 dimerisation (LB)
FC33/pSDC115 contains plasmid with two cer-site (to be used in week 3)
DS956 and DS957 (to be used in week 3 or 4)
Monday 16th
Morning:
BEFORE 12: prepare things to be autoclaved
Prepare basic solutions and set up the bench
– 400 ml liquid LB (to be split in flasks and universal bottles)
– 10ml of 1000X Ampicillin stock (concentration 100mg/ml) to be filtered using a syringe with a 0.2 μm filter and divided in 1ml aliquots in Eppendorf tubes. Store in freezer -20°C.
– 100 ml of sterile ddH20 (to be autoclaved)
– 100 ml of 1M CaCl2 solution
– 100ml of 800mM MgCl2 solution
Prepare plates of required E.coli strains (from freezer stocks) – incubate at 37°C ON
– DS941
– DS945
– DS956
– DS957
– FC33
Afternoon:
Prepare liquid inoculum of DS941 cells for ON growth (in one 20ml Universal bottle) and incubate at 37C, 140rpm
Tuesday 17th
Morning:
Make DS941 competent cells
Wednesday 18th
Morning:
Prepare 250 ml LB/agar (to be autoclaved – keep going in the afternoon)
Resuspend dry DNA (G-block) in Xμl (volume depending on the specific tube, probably 15μl) and leave at room temperature on the bench for at least 1h
Measure concentration of pUC19
Restriction digestion HindIII and EcoRI (all except for Adil, who has to work with KpnI and HindIII)
Set up three reactions for pUC19:
– Just HindIII (2 μl buffer + 5 μl pUC19 + 1 μl HindIII +12 μl water)
– Just EcoRI (Adil: just KpnI) (2 μl buffer + 5 μl pUC19 + 1 μl EcoRI + 12 μl water)
– HindIII+EcoRI (2 μl buffer + 15 μl pUC19 + 1 μl HindIII + 1 μl EcoRI + 1 μl water)
Set up one incubation for the G-block
– Double digest with HindIII+EcoRI (Adil: KpnI+HindIII) (1 μl buffer + 5 μl DNA + 1 μl of diluted HindIII + 1 μl of diluted EcoRI + 2 μl water)
Incubate at 37 degrees for 1h, then heat inactivate at 65degrees for 10min. Load the digested plasmid on gel to gel-purify the double digested product (load 5 μl of the non-digested plasmid as control)
While the restriction reaction is incubating at 37 degrees, pour a 0.8% agarose gel – DO NOT ADD GEL RED!
Prepare the Azure A solution
When the restriction is completed, load the samples on the agarose gel and run the electrophoresis
Afternoon:
When the LB/agar has cooled enough to hold in hand, add 250ul of ampicillin and pour in ~20 plates
Stain the gel with Azure A
Cut and elute the band corresponding to the restricted plasmid
Start the ligation reaction
Thursday 19th
Transformation DS941/ligation
Prepare LB/Amp plates (if not done already)
Friday 20th
Observe the results on the plates
Preparation of agarose gels
To prepare a 0.8% agarose gel, measure 0.4g of agarose and dissolve it in 50 ml of gel running buffer (TBE) – to melt the agarose, microwave 1min at power 5 (WEAR THE GLOVE when using the microwave) and stir the bottle, microwave for 1min at power 5, stir and keep microwaving in short 15s cycles (power 5) until completely molten. If you need to elute a DNA band from gel – you do not need to add any dye to the gel because you will stain your gel with Azure A after the run is finished. If you only need to observe the gel – add an aliquot of GelRed, available at the help desk. Pour the molten agarose into the gel apparatus (make sure end plates and comb are firmly in place). Leave to set on the bench. When the agarose is set, pour 50ml gel running buffer (TBE) onto the top of the gel. Remove the end plates and comb and stand the gel on a sheet of black plastic, close to the power pack. Do not move the gel once you have started loading your sample.
Gel electrophoresis
To prepare your samples, add 5 μl of Orange DNA Loading Dye (6x) buffer (gel loading buffer) to each sample and mix gently by flicking or pipetting. Using a 20 μl pipette collect 20 μl of sample. Load the 20μl sample into the required well of the gel. Use the central wells for the samples. Load 5 μl of “1kb Plus DNA ladder” into the outer wells of the gel. Connect the gel to the power pack and run at 70V (if running 2 gels at once, still use 70 volts). However, you may have to increase the current setting to allow the gel powerpack to reach 70 volts. Ask if not sure. Check for bubbles to make sure the gel is actually running! Run the samples until the stain in the loading buffer almost touches the end of the gel and then switch off the power supplier before removing the lid of the electrophoresis apparatus. You are now ready to stain your gel in Azure A.
Preparation of 2X Azure A solution (see http://2015.igem.org/Team:Glasgow/AzureA)
To prepare 500 mL of 2x Azure A (0.08% Azure A/40% Ethanol):
Dissolve 0.4g Azure A chloride solid in 500mL 40% Ethanol. Store in a glass screw capped bottle out of direct sunlight, or alternatively wrap the bottle in aluminium foil. Azure A will bleach over time in sunlight and lose effectiveness.
Prior to use, a 1x (0.04% Azure A/20% Ethanol) solution should be prepared by diluting the 2x stock 1:1 with distilled or deionised water to a desired final volume. 1x stock should also be stored in a glass screw capped bottle out of direct sunlight.
Stain with Azure A
Pour off the buffer solution and move the gel in the plastic box. Put on some gloves to prevent the stain from touching your skin. Pour enough 1x Azure A solution (0.04% Azure A in 20% ethanol) into the container to completely cover the gel. Shake the container for 10-15 minutes by hand, then return the stain to a bottle for re-use. (The DNA stain can be re-used several times before you need to replace it. However, you may find that older stain needs to be left on the gel for a little longer than 10 minutes). After a 10-15min stain, DNA bands of high concentration may already be visible at this stage, but I’d recommend at least one round of destaining for improved visualisation. To destain, cover the gel in distilled/deionised water. Shake for 10-15 mins. The destaining solution should be discarded into a sink drain along with running water. Repeated rounds of destaining will improve visualisation bands of weak concentration.
Gel extraction with Azure A
Gel extraction after staining with Azure A follows the same protocol as an Ethidium bromide gel extraction (follow the instructions in the QIAquick Gel Extraction Kit by QIAGEN), without the need to use a UV transilluminator and associated face protection. The only difference caused by use of Azure A is a change of colour not normally seen in use of the kit; after addition of yellow Buffer QG for the fragment melting stage the colour changes to green, due to the interaction of the blue and yellow dyes in the gel fragment and QG respectively – this is inconsequential, no need to worry about it.
N.B. This is not the same erroneous colour change that is warned about in the protocol due to issues with pH.
Preparation of LB broth and LB plates (watch Lauren’s videos on Aula – you may want to autoclave a series of small universals for your experiments, but you will also need 100ml in conical flasks for each set of competent cells). Instructions are written on the side of the LB or LB/agar bottle. Make sure you add your name and my initials on the autoclave tape so that the technicians will know whose bottles they are working with. For some of your experiments you will need to add ampicillin to your LB plates, and this needs to be done when the LB/agar is still liquid but has cooled down enough after being autoclaved (you know this is the case when the bottle is still hot, but you can hold it in your hands without burning them). Your antibiotic stock will be 1000 times more concentrated than your final concentration, so you will need to add 1/1000 of the volume (for example, 1ml of antibiotic in 1l of LB). Make sure you stir the bottle after adding the ampicillin before you pour the plates.
Ampicillin stock
You need to prepare a 1000X Ampicillin stock 100mg/ml, which you will then sterilise using a 0.2nm filter. To prepare this solution, weigh 0.5g of ampicillin in a 15ml tube on the scale and dissolve it in 5ml of distilled water. Use a vortex to stir the solution until all powder has been solubilised. Filter the solution in a new 15ml tube using a syringe and a 0.2μm filter. Divide the solution in five 1ml aliquotes in sterile eppendorf tubes and store at -20degrees when not needed.
Set up of ON liquid cultures
Preparation of competent cells:
Inoculate a 5 ml overnight-culture in LB-medium without antibiotics over night. In the morning, inoculate 50 ml LB-medium with 0.5ml of the overnight culture and incubate to an OD600 = 0.4 – 0.5 at 37 °C and 140 rpm. Transfer cells to two 50 ml sterile falcon tubes (add 25ml of cells in each) and incubate on ice for at least 20 min. From this step on, keep cells always cold. Centrifuge for 10 min at 2700 rpm and 4 °C (pre-cool centrifuge) and discard the supernatant. Carefully suspend each pellet in 25 ml of icecold CaCl2-MgCl2 solution (80mM MgCl2-20mM CaCl2). Centrifuge for 10 min at 2700 rpm and 4 °C and discard the supernatant. Carefully suspend each pellet in 10 ml icecold 100 mM CaCl2 solution, and combine them in a single tube (final volume will be 20ml). Centrifuge for 10 min at 2700 rpm and 4 °C and discard the supernatant. Carefully suspend the pellet in 500 μl icecold 100 mM CaCl2 solution, then you can use the cells for transformation. To store the competent cells: After last resuspension step, add one volume of sterile ice-cold 50% glycerol to two volumes of cells (ex. add 500μl 50% glycerol to 1ml cells) and gently mix by pipetting up and down. Split the cells in several 100ul aliquotes in sterile labelled eppendorf tubes, pre-cooled in ice. Store in ULT freezer.
Transformation of E.coli cells
When you do a transformation with a plasmid of interest, you will always need to have a positive and a negative control. Your positive control is the empty plasmid pUC19, your negative control a transformation done with water instead of DNA. If you are doing multiple transformations at the same time, you will only need to only need to have one positive and one negative control.
Restriction digestion HindIII and EcoRI
Set up three reactions for pUC19:
Just HindIII (2 μl buffer + 5 μl pUC19 + 1 μl HindIII +12 μl water)
Just EcoRI (2 μl buffer + 5 μl pUC19 + 1 μl EcoRI + 12 μl water)
HindIII+EcoRI (2 μl buffer + 15 μl pUC19 + 1 μl HindIII + 1 μl EcoRI + 1 μl water)
Set up one incubation for the G-block
Resuspend the dry DNA in 15 μl of sterilised water and leave at room temperature on the bench for at least 1h.
Double digest with HindIII+EcoRI (1 μl buffer + 5 μl DNA + 1 μl of diluted HindIII + 1 μl of diluted EcoRI + 2 μl water)
Incubate at 37 degrees for 1h, then heat inactivate at 65degrees for 10min. Load the digested plasmid on gel to gel-purify the double digested product (load 5 μl of the non-digested plasmid as control)
Ligation protocol
https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202
(see here for calculations http://nebiocalculator.neb.com/#!/ligation)
You will need to set up this reaction:
2 μl ligase buffer + 8 μl purified HindIII/EcoRI pUC19 + 8 μl HindIII/EcoRI insert + 1 μl water + 1 μl T4 ligase
Leave at room temperature for 1h (or longer, even over night), then heat inactivate at 65degrees for 10min in the heat block