WET-LAB PROTOCOLS

Preparation of LB-Antibiotic Plates

Restriction Digest Protocol for BioBricks

QIAGEN Gel Extraction Protocol

Ligation Protocol

Heat-Shock Transformation Protocol

Colony PCR Protocol

QIAGEN Plasmid Prep Protocol

M9 Minimal Media Protocol

Agarose Gel Electrophoresis Protocol

-80°C stock preparation Protocol

Mutagenesis PCR Protocol

Alkaline Phosphatase Protocol

Glycogen Extraction Protocol

Debranching Glycogen Protocol

Starch Quantification Protocol

Electroporation Transformation

Agrobacterium tumefaciens-mediated infiltration of Nicotiana benthamiana

One-Step Golden Gate Digestion-Ligation Protocol

Preparation of LB-Antibiotic Plates

1. Dissolve 200 mL LB agar in microwave (Power 5, 5 mins).

2. Allow to cool on bench.

Under aseptic conditions:

3.Add antibiotics to the desired final concentration.

4. Pour plates. Label edges first.

5. Allow to set near to flame, leave gap for condensation.

6. Store in the fridge.

Restriction Digest Protocol for BioBricks

Add 2 µL restriction enzyme buffer H ×10 to a sterile 0.6 mL tube. Add 0.5-1 µg of DNA. Add appropriate amount of deionized H2O to bring volume of entire digest once enzymes have been added, to 20 µL. Add 0.5 µL of restriction enzyme (EcoR1, Spe1, Xba1 or Pst1). Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of the tip. To ensure only 0.5 µL of enzyme is added, just touch the tip to the surface of the liquid when pipetting. Incubate for 2 hours at 37ºC. QIAGEN Gel Extraction Protocol Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Weigh the gel slice in a colourless tube. Add 3 volumes of Buffer QG (Solubilization buffer) to 1 volume. For >2% agarose gels, add volumes Buffer QG. Incubate at 50ºC for 10 minutes. Vortex the tube every 2-3 minutes to help dissolve the gel. After the gel slice has dissolved completely, check that the colour of the mixture is yellow. If the colour of the mixture is orange or violet, add 10 µL of 3 M Sodium Acetate, pH 5.0, and mix. The colour of the mixture will turn yellow. Add 1 gel volume of isopropanol to the sample and mix. 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 1 minute at 17,900 × g (13,000 rpm). Discard the flow-through and place the QIAquick column back into the same tube. Add 0.5 mL Buffer QG to the QIAquick column and centrifuge for 1 minute at 17,900 × g (13,000 rpm). Discard the flow-through and place the QIAquick column back into the same tube. To wash, add 0.75 mL Buffer PE (Wash Buffer) to QIAquick column and centrifuge for 1 minute at 17,900 × g (13,000 rpm). Discard flow-through and place the QIAquick column back into the same tube. Centrifuge the QIAquick column once more in the provided 2 mL collection tube for 1 minute at 17,900 × g (13,000 rpm) to remove residual wash buffer. Place QIAquick column into a clean 1.5 mL microcentrifuge tube. Add 30 µL Buffer EB (Elution Buffer) to the centre of the QIAquick membrane, let the column stand for 1 minute, and then centrifuge for 1 minute at 17,900 × g (13,000 rpm). Ligation Protocol Add 8.5 μL of insert and plasmid vector in a specific ratio. The insert to vector ratios commonly used were 3:1, 6:1, and 10:1. Add 1 μL of T4 ligase 10x buffer. Add 0.5 μL of T4 ligase. Incubated at 16°C overnight, 4° over the weekend or 30 minutes at room temperature. Heat-Shock Transformation Protocol Thaw competent E. coli cells on ice. For DH5α cells, add 10 µL of plasmid DNA to 50 µL of competent cells. For BL21 cells, add 1 µL of plasmid DNA (1 pg – 100 ng) to 50 µL of competent cells. Mix by resuspending solution 4-5 times. Do not vortex. Incubate on ice for 30 minutes. Do not vortex. Heat shock at 42ºC for 2 minutes. Incubate on ice for 2 minutes. Add 1 mL of LB (no antibiotic). Incubate at 37ºC for 2 hours with shaking (200 rpm). Spin in centrifuge for 1 minute at 17,900 × g (13,000 rpm). Plate out 100 µL of culture onto a plate with the relevant antibiotic. Grow at 37ºC overnight or at room temperature over the weekend. Colony PCR Protocol Prepare a master-mix appropriate to the number of samples as follows (add Taq polymerase last). For 15 PCR samples, the amounts used were as follows: 37.5 µL of Taq Polymerase ×10 buffer, 3.75 µL 200 µM dNTP mix, 255 µL deionized H2O, 30 µL 3.125 pmol/µL VF2 primer, 30 µL 3.125 pmol/µL VR primer, 3.75 µL Taq Polymerase. Aliquot into PCR tubes. Label new agar plate (with same antibiotic) with area for each colony. Choose colony, touch on new agar plate, and add to mastermix. Controls: Water (negative) and plasmid vector with a different-size insert (positive). PCR conditions 95ºC for 15 minutes 94ºC for 30 seconds 62ºC for 30 seconds 68ºC for 3.5 minutes Repeat steps 2-4 39 times 68ºC for 20 minutes 4ºC forever QIAGEN Plasmid Prep Protocol Pellet 2 mL of a bacterial overnight-culture by centrifugation at >8000 rpm (6,800 × g) for 3 minutes at room temperature (15-25ºC). Repeat Step 1. Resuspend pelleted bacterial cells in 250 µL Buffer P1 (Resuspension Buffer) and transfer to a microcentrifuge tube. Add 250 µL Buffer P2 (Lysis Buffer) 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 minutes. Add 350 µL Buffer N3 (Neutralization Buffer) and mix immediately and thoroughly by inverting the tube 4-6 times. Centrifuge for 10 minutes at 17,900 × g (13,000 rpm). Apply the supernatant from Step 6 to the QIAprep spin column by pipetting. Centrifuge for 60 seconds at 17,900 × g (13,000 rpm) and discard the flow-through. Wash the QIAprep spin column by adding 0.5 mL Buffer PB (Binding Buffer). Centrifuge for 60 seconds at 17,900 × g (13,000 rpm) and discard the flow-through. Wash the QIAprep spin column by adding 0.75 mL Buffer PE. Centrifuge for 60 seconds at 17,900 × g (13,000 rpm) and discard the flow-through. Transfer the QIAprep spin column to the collection tube. Centrifuge for 1 minute to remove residual wash buffer. Place the QIAprep column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 30 µL Buffer EB (10 mM Tris.Cl, pH 8.5) to the centre of the QIAprep spin column, let stand for 1 minute, and centrifuge for 1 minute. M9 Minimal Media Protocol Add the following materials to a 1 L flask; 6 g Na2HPO4 3 g KH2PO4 0.5 g NaCl 0.222 g (NH4)2SO4 Adjust the pH to 7.4 and make up to 1 litre with water and autoclave. To the above, under sterile conditions, add; 650 µL Trace Elements Stock (composition below), 6 mL 50% Glucose 1 mL 1% filter-sterilised Thiamine 1 mL 1 M MgCl2 0.1 mL 1 M CaCl2 Trace Element Stock Composition 500 mg EDTA Adjust pH to 8.0 and add, 550 mg CaCl2.2H2O 140 mg MnSO4.H2O 27 mg CuCl2 220 mg ZnSO4.7H2O 45 mg CoCl2.6H2O 26 mg Na2MoO4.2H2O 40 mg H3Bo4 26 mg KI Adjust pH to 8.0 then add, 375 mg FeSO4.7H2O Adjust pH to 8.0 again and filter sterilise. Agarose Gel Electrophoresis Protocol To prepare a 1% agarose solution, place 5 g of agarose into 500 mL of TAE buffer and dissolve in microwave (Power 5, 10 minutes). To prepare a 2% agarose solution, place 10 g of agarose into 500 mL of TAE buffer and dissolve in microwave (Power 5, 10 minutes). Store in 50°C oven. For small gels, place 30 mL of agarose and 15 μL of Ethidium Bromide into a small gel tray. For larger gels, place 200 mL of agarose and 100 μL of Ethidium Bromide into a large gel tray. Insert well mold and allow gel to cool and set. Place tray into electrophoresis apparatus and remove well mold. Add 4 μL of Loading Buffer (x6) to all samples. Add 2 μL of Ladder to extremity wells and fill wells with 24 μL of samples. For small gels, run gel at 95 V for 30 minutes. For larger gels, run gel at 95 V for 2 hours. -80°C stock preparation Protocol Place 952 μL of liquid culture into an eppendorf tube. Add 75 μL of dimethyl sulfoxide and mix. Store in -80°C freezer. Mutagenesis PCR Protocol Prepare the PCR samples, in PCR tubes, as follows; 4µL HF ×5 Buffer 0.25 µL 20 mM dNTPs 2 µL 3.125 µM Forward Modification Primer 2 µL 3.125 µM Reverse Modification Primer 50 ng of DNA Deionized H2O to bring total volume up to 25 μL 0.5 µL Phusion polymerase PCR Conditions 95°C for 15 minutes. 94°C for 30 seconds. 72°C for 3.5 minutes. Repeat Step 3 for 34 cycles. 72°C for 20 minutes. 4°C forever. Alkaline Phosphatase Protocol Vortex all reagents before use. Place all of the linearized vector from a restriction digest in an eppendorf tube. Add 2 μL of rAPid alkaline phosphatase x10 buffer. Add 1 μL rAPid alkaline phosphatase. Add deionized water to make the final volume up to 20 μL. Incubate at 37°C for 60 minutes. Heat-inactivated at 75°C for 2 minutes. Glycogen Extraction Protocol Harvest E. coli cells from liquid cultures by centrifugation at 5000 rpm for 10 minutes and resuspended in 10 mL water in a 50 mL Falcon tube. Pellet resuspended cells by spinning at 5,000 × g for 10 minutes. Discard supernatant and resuspend pellet in 10 mL fresh water. Sonicate at room temperature at 10 micron amplitude for 3 minutes, 1 second on and 2 seconds off. Transfer to 50 mL centrifuge tubes and centrifuge at 30,000 × g for 15 minutes. Transfer supernatant to a 50 mL Falcon tube. Add 5 mL of 0.2 M glycine, pH 10.5 and 5 mL chloroform. Shake vigorously and spin at 2000 rpm for 3 minutes to separate into aqueous and organic layers. Transfer top, aqueous layer to a new 50 mL Falcon tube with a pipette and repeat step 5. Transfer top, aqueous layer to a round-bottomed flask and remove any remaining chloroform using rotary evaporation. Transfer to a 30 kDa spin filter and concentrate to ~8 mL by spinning at 5000 × g for approximately 55 minutes. Check after 30 minutes of centrifugation. Transfer to 8 × 1 mL ultracentrifuge tubes and balance all to within 1 mg of each other. Spin in Ultracentrifuge at 108,000 × g (55,000 rpm) at 4ºC for 2-3 hours. Discard supernatant and resuspend pellets in 2 mL total volume of water and add to 50 mL Falcon tube. Precipitate glycogen with 8 mL cold ethanol. Spin at 4,000 × g for 10 minutes and discard supernatant. Dissolve pellet in 2 mL of water and freeze-dry overnight to yield the glycogen as an amorphous white powder. Debranching Glycogen Protocol 1 mg of glycogen sample was dissolved in 50 μL of 100 mM sodium acetate buffer, pH 4.0. 2 μL of isoamylase was added. Incubated at 37°C for 2 hours. Terminate reaction by heating at 95°C for 5 minutes. Precipitated with 125 μL ethanol and dissolved in 50 μL deionized water. Freeze-dried overnight to yield an amorphous white powder. Starch Quantification Protocol Harvest plant material (0.2-0.5 g) into capped tubes and freeze immediately in liquid nitrogen. Once tissue is frozen, tubes can be transferred to dry ice. If starch content is to be expressed on a fresh-weight basis, sample weight can be determined either by weighing very rapidly prior to freezing, by re-weighing the sample-containing collection tubes, or by weighing out part of the frozen material for analysis. If starch content is to be expressed on a dry-weight basis, the sample can be freeze-dried and then weighed. To obtain a robust estimate of the starch content of a particular tissue, it will normally be necessary to collect and analyse several biological replicates. Transfer tissue to a tube containing 5 mL 80% aq. vol/vol ethanol and incubate in a boiling water bath for 3 minutes. Spin samples in a benchtop centrifuge at ≥3,000 x g for 5-10 minutes at room temperature, to pellet solids and discard supernatant. Repeat this ethanol extraction twice more, discarding the supernatants. Allow ethanol to evaporate from the final pellet. Transfer pellet to a mortar with a minimum volume of water and homogenise thoroughly to a smooth consistency. Transfer to a volumetric container and make up to 5 mL with water washings from the mortar. Where large numbers of samples are to be processed, ethanol-extracted pellets can be homogenised in small volumes of water in a multi-tube ball mill. Add 0.5 mL of the homogenate to each of four tightly sealing screw-capped microcentrifuge tubes. After sealing, heat to 100°C for 10 minutes to gelatinize starch granules. Alternatively, samples can be autoclaved at 120°C. Allow to cool. Add 0.5 mL 200 mM sodium acetate, pH 5.5, to each tube. To two of the tubes add roughly 6 units of α-amyloglucosidase and at least 0.5 units of α-amylase. Add a solution of equivalent volume and composition without the enzymes to the other two tubes (control samples). Incubate tubes at 37°C for 4 hours. Spin tubes in microcentrifuge at ≥10,000 x g for 5 minutes at room temperature to remove particulate material. Assay samples of the supernatant for glucose. Several assays for glucose are available. Record OD340 and leave the cuvette in the spectrophotometer. Place a minimal volume of a mix of 1 unit hexokinase and 1 unit of glucose 6-phosphate dehydrogenase onto a plastic microspatula and mix rapidly with the contents of the cuvette without removing it from the spectrophotometer. The OD340 is recorded again after a constant value is reached. This should take no more than 10 minutes. Calculate the starch content content of the tissue. Electroporation Transformation Protocol E. coli Remove 50 µL of electrocompetent E. coli cells from the -80°C freezer and thaw on ice. Add 5 µL of DNA and transfer to a clean 1m electroporation cuvette. Electroporate cells at 1.8kV using a Bio-rad micropulser machine. Add 500 µL of LB broth to the electroporated cells and transfer to a clean 1.5 mL Eppendorf tube. Incubate at 37°C with shaking for 1 hour. Spread plate 100 µL onto plates containing the relevant antibiotics and incubate overnight at 37°C. Agrobacterium tumefaciens Remove 50 µL of electrocompetent Agrobacterium GV3101 cells from the -80°C freezer and thaw on ice. Add 5 µL of DNA and transfer to a clean 1m electroporation cuvette. Electroporate cells at 1.8kV using a Bio-rad micropulser machine. Add 500 µL of LB broth to the electroporated cells and transfer to a clean 1.5 mL Eppendorf tube. Incubate at 28°C with shaking for 1 hour. Spread plate 100 µL onto plates containing the relevant antibiotics and incubate overnight at 28°C. Agrobacterium tumefaciens-mediated Infiltration of Nicotiana benthamiana From a single Agrobacterium colony or glycerol stock inoculate 5 mL LB media containing the appropriate antibiotic(s)*. Grow overnight at 28 °C, 250-300 rpm. Pellet by centrifugation at RT (19-23 °C), 4000 rpm for 15 minutes. Resuspend in 2% sucrose, 1% Murashige and Skoog (MS) salt, 150 μM acetosyringone. Incubate at RT (19-23 °C) for at least 3 hours. Infiltrations are made by injecting the culture to the underside of a leaf with a blunt syringe. Plants are left in a growth room at a temperature around 19-23 °C during co-incubation of the Agrobacterium and the plant. Ones-Step Golden Gate Digestion Ligation Protocol 1. Add ATP to buffer that will be used, as it is required for the T4 Ligase to work. 2. Decide which buffer to use, restriction or ligase buffer. The protocol will differ depending on the buffer. BpiI is required to make level 0 constructs. 3. To make level 0 parts using the ligase buffer, the following were added: 100-200ng of acceptor plasmid, plasmids containing each module to be inserted in a 2:1 ratio of insert: acceptor, 1.5µL of T4 Ligase buffer with 200 units of the T4 DNA ligase, 1.5µl of Brovine Serum Albumin (10x) and 5 units of BpiI. They are then put in the following conditions: 20 seconds 37°C, (3 minutes 37°C, 4 minutes 16°C) X26, 5 minutes 50°C, 5 minutes 80°C, 5 minutes 16°C. 4. To make level 1s, the type of buffer must again be decided. Using the ligase buffer will follow the same conditions as the level 0, but BsaI will be used. 5. To make level 2s, repeat step 3 with the products of step 4 (optional) 6. Use 5µl of the one-pot dig-lig reaction to transform electrocompetent E.coli cells. 7. Select positive clones using LB agar with appropriate antibiotics. 8. Decide on the primers used for amplification and/or sequencing.