Difference between revisions of "Team:Linkoping Sweden/Protocols"

Agarose gelelectrophoresis

Material

• Agarose
• 10x TBE buffer
• Loading dye
• Molecular weight marker

Procedure

Agarose gel

1. Dilute 10 ml 10x TBE buffer with water to an final volume of 100 ml. 2. Add 0,5-2,0 g agarose depending on the size of your fragment. 3. Heat the agarose in microwave until completely liquid. Let mixture cool until approximately 60℃ and pour agarose gel. Let cool for at least 30 minutes.

DNA separation

1. Mix 4 μl loading dye with 5 μl DNA and 11 μl H2O (and conntrol without DNA). 2. Add agarose gel in electrophoresis apparature and fill with 1x TBE buffer to cover the gel. 3. Add 5 μl premixed molecular weight marker and 20 μl of DNA mixture or control to separate wells. 4. Separate the DNA fragments by applying 150 Volts 5. Stain DNA fragments with ethidium bromide (15 ul in approximately 400 1x TBE) for 20 minutes. 6. Wash in dH2O for 5 minutes 7. Illuminate with 300-360 nm, photograph gel and estimate size or amount of DNA

CULTIVATION OF ALGAE

Material

• 100 ml TAP – medium
• 1ml Alg suspension

Procedure 1. Mix 100 ml TAP – medium with 1 ml alg suspension 2. Incubate the culture on a shaker (or pump if available) (100 - 150 rpm, LED light 14 hours/day, temperature constant 22 °C) 3. Measure the growth using a spectrometer (750 nm) every day about the same time. Incubate until the growth curve reaches 0.5.

CULTIVATION OF ALGAE IN THE DARK

Material

• 100 ml TAP – medium
• 1 ml Alg suspension

Procedure 1. Make a new TAP - medium with 3.4 g/l acetate instead of 3.2 ml/l acetic acid 2. Mix 100 ml of the new TAP – medium with 1 ml alg suspension 3. Incubate the culture on a shaker (or pump if available) (100 - 150 rpm, completely in the dark, temperature constant 22 °C) 4. Measure the growth using a spectrometer (750 nm) every day about the same time. Incubate until the growth curve reaches 0.5.

DIGESTION

Material

• 1 μl Restriction enzyme
• DNA
• 1 μl 10 X CutSmart buffer
• Autoclaved H2O To a final volume of 10 μl

Procedure 1. Mix restriction enzyme, DNA, Cutsmart buffer and autoclaved H2O. 2. Incubate the restriction digest at 37 °C for 1 h 3. Incubate at 80 °C for 20 min for heat inactivation

LIGATION

Material

• 50 ng Linearized Vector
• 3X molar excess Gene Fragment
• 10 μl 2X Quick Ligase Buffer
• 1 μl Quick DNA Ligase
• Autoclaved H2O To a final volume of 21 μl

Procedure 1. Combine 50 ng of vector with a 3-fold molar excess of insert. Use NeBioCalculator to calculate molar ratios. Adjust volume to 10 μl with autoclaved H2O. 2. Add 10 μl of 2X Quick Ligation Buffer and mix. 3. Add 1 μl of Quick T4 DNA Ligase and mix thoroughly. 4. Centrifuge briefly and incubate at room temperature (25 °C) for 5 min. 5. Chill on ice, then transform or store at -20 °C.

Note: Do not heat inactivate the ligase. Heat inactivation dramatically reduces the transformation efficiency.

E. coli Calcium Chloride competent cell

1. Inoculate a single colony into 5ml LB in a 50ml Falcon tube. Grow overnight at 37°C. 2. Use 1ml to inoculate 100ml of LB in 250ml bottle the next morning. 3. Shake at 37°C for 1.5-3hrs. OR Inoculate a single colony into 25ml LB in a 250 ml bottle in the morning. and then shake at 37°C for 4-6 hrs. 4. Put the cells on ice for 10 mins (keep cold form now on). 5. Collect the cells by centrifugation for 3 mins at 6000 rpm. 6. Decant the supernatant and gently resuspend on 10 ml cold 0.1M CaCl (cells are susceptible to mechanical disruption, so treat them nicely). 7. Incubate on ice for 20 mins 8. Centrifuge for 3 minutes at 6000 rpm. 9. Discard supernatant and gently resuspend on 5ml cold 0.1 M CaCl2/15% Glycerol 10. Dispense in microtubes (300μl/tube). Freeze in - 80°C.

Transformation of Ca2+ competent cells 1. Put 1μl of circular plasmid or all of a ligation reaction of plasmid DNA in a microtube. Gently add ~100μl of competent cells. Do NO DNA control tube with cells and no DNA. 2. Incubate for 30 mins on ice. 3. Heat shock for 2 mins at 42°C. Put back on ice. 4. Add 900 μl of LB to tubes. Incubate at 37°C for 30 mins. 5. Plate 100-1000 μL of the cells in LB-Amp or LB-Carb (100μg/ml) plates. Plate 100 μl of the NO DNA control in a blood plate (to check for quality of cells). Grow overnight. You can save the rest in -80°C with 15% of Gly in case there would not be any colonies. 6. If you need a lot of colonies or the ligation is of low efficiency, centrifuge the transformation for 1 min at 8000 rpm, discard 900 μL of supernatant, resuspend on the 100 μL left and plate the whole lot.

CaCl2/15% Glycerol – solutions for competent cells Material

• 0.1 M CaCl2
• 15 % glycerol solution

Procedure 1. Mix 1 ml 1 M CaCl2 solution with 1.5 ml 100 % glycerol and 7.5 ml dH2O.

GLYCEROL STOCK 1. After you have bacterial growth, add 500 μL of the overnight culture to 500μL of 50% glycerol in a 2 mL snap top tube and gently mix. Note: Make the 50% glycerol solution by diluting 100% glycerol in dH20. 2. Freeze the glycerol stock tube at -80°C. The stock is now stable for years, as long as it is kept at -80°C. Subsequent freeze and thaw cycles reduce shelf life.

PCR

Material

• Standard Taq Reaction Buffer (10X) 5 μl
• Deoxynucleotide Solution Mix 1 μl
• Forward primer (10 μM stock solution) 1 μl
• Reverse primer (10 μM stock solution) 1 μl
• DNA, plasmid 1 pg- 1ng
• Taq DNA polymeras (diluted)* 1 μl
• Nuclease free water Bring reaction voulme to 50 μl

Procedure

1. *Dilute stock Taq DNA polymeras 1:4 in 1X Standard Taq Reaction Buffer 2. Prepare reaction mixture. Add Taq DNA polymeras just before starting PCR-reaction. 3. PCR-reaction

Initial denaturation 98 C 30 seconds 25 cycles Denaturation 95 C 15-30 seconds Annealing 45-68C 15-60 seconds Extension 68 C 1 minute/kb Final extension 72 C 2 minutes Hold 4 C

New England Biolab’s TM calculator can be used to estimate annealing temperature: http://tmcalculator.neb.com/#!/

PLASMID PREPARATION

Harvest cells: Pellet 1–5 ml of an overnight recombinant E. coli culture by centrifugation. Transfer the appropriate volume of the recombinant E. coli culture to a microcentrifuge tube and pellet cells at ≥12,000 3 g for 1 minute. Discard the supernatant.

1. Resuspend cells: Completely resuspend the bacterial pellet with 200 µl of the Resuspension Solution. Vortex or pipette up and down to thoroughly resuspend the cells until homogeneous. 2. Lyse cells: Lyse the resuspended cells by adding 200 µl of the Lysis Solution. Immediately mix the contents by gentle inversion (6–8 times) until the mixture becomes clear and viscous. Do not vortex. Harsh mixing will shear genomic DNA, resulting in chromosomal DNA contamination in the final recovered plasmid DNA. Do not allow the lysis reaction to exceed 5 minutes. 3. Neutralize: Precipitate the cell debris by adding 350 µl of the Neutralization/Binding Solution. Gently invert the tube 4–6 times. Pellet the cell debris by centrifuging at ≥12,000 x g or maximum speed for 10 minutes. If the supernatant contains a large amount of floating particulates after centrifugation, recentrifuge the supernatant before proceeding to step 6. 4. Prepare Column: Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tube, if not already assembled. Add 500 µl of the Column Preparation Solution to each miniprep column and centrifuge at ≥12,000 x g for 30 seconds to 1 minute. Discard the flow-through liquid. 5. Load cleared lysate: Transfer the cleared lysate from step 3 to the column prepared in step 4 and centrifuge at ≥12,000 x g for 30 seconds to 1 minute. Discard the flow-through liquid. 6. Wash column: Add 750 µl of the diluted Wash Solution to the column. Centrifuge at ≥12,000 x g for 30 seconds to 1 minute. Discard the flow-through liquid and centrifuge again at maximum speed for 1 to 2 minutes without any additional Wash Solution. 7. Elute DNA: Transfer the column to a fresh collection tube. Add 50 µl water to the column. Centrifuge at ≥12,000 x g for 1 minute. The DNA is now present in the eluate and is ready for immediate use or storage at –20 °C.

Transformation

Material

• E.coli XL1-blue supercompetent cells 20 μl
• DNA 1 μl (5 ng)
• Soc-medium 100 μl
• LB-agarplates with Chloramphenicol (CHL) resistence

Procedure

1. Thaw the E.coli XL1-blue supercompetent cells on ice 2. Portion in 20 μl and add approximately 1 μl (5 ng) DNA 3. Mix gently with pipette tip and incubate on ice for 30 minutes 4. Heat shock cells in heating block 45 seconds at 42℃, then place on ice for 2 minutes 5. Add 100 μl preheated (42℃) Soc-medium and incubate the transformation mixture for 2 h at 37℃ 6. Spread all of the transformation mixture (121 μl) on preheated agarplates (37℃) with Chloramphenicol resistance 7. Incubated over night (at least 16 hours). Count the number of colonies 8. Store in cold room, approximately 4℃

NOTE Control plate is done without DNA in order to control the antibiotic resistance works