Describe the research, experiments, and protocols you used in your iGEM project.
From https://2016.igem.org/Team:Linkoping_Sweden/Protocols#Cultivation_of_algae
Material
1. 100 ml TAP – medium
2. 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)
Measure the growth using a spectrometer (750 nm) every day about the same time. Incubate until the growth curve reaches 0.5 (6).
Transformations (Electroporation or Heat Shock)
To thaw:
1. Competent cells (on ice) … Found in -80 freezer
2. SOC medium (@ room temp.) … Found in -80 freezer
3. Turn on shaking incubator
4. Turn on water bath (Heat shock only)
5. Agar plates in incubator
The DNA (@ room temp)
Electroporation
1. Combined 40 μL of electrically competent DH5a cells and 1 μL of ligated DNA to an Eppendorf tube.
2. Transferred the contents of the Eppendorf tube to a cuvette and lightly tapped the cuvette on the table to evenly distribute the contents and to get rid of air bubbles.
3. Placed the cuvette into the Bio-Rad MicroPulser and delivered the electric shock.
4. Immediately after, added 900 μL SOC medium to the cuvette and micropipette mixed the solution.
5. Transferred the solution from the cuvette to a shaker tube and placed in the shaker at 37°C at 200 rpm for 1 hour.
6. After shaking for 1 hour, streaked 150 μL of the solution onto an agar plate with the respective antibiotics.
7. Incubated plates at 37°C for at least 24 hours.
Heat Shock
1. Thawed One Shot TOP10 chemically competent cells on ice (50 ul).
2. Added 2 μL of DNA sample into competent cells
3. Incubated the cells on ice for 35 minutes.
4. After the ice incubation, placed the samples into a 42° C water bath for 30 seconds.
5. Quickly took them out and immediately added 250µL of SOC medium
6. Place the samples into a 37° C shaking water incubator for 1 hour at 200 rpm.
7. After shaking for 1 hour, streaked 150 μL of the solution onto an agar plate with the respective antibiotics.
8. Incubated plates at 37°C for at least 24 hours.
Preparing, Loading, and Running a 1% Agarose Gel
Things to Thaw:
1. MW ladder (not on ice) … found in freezer
2. Loading Dye (not one ice) … found above scale
Preparing
1. Added 1 g of Agarose in 100 mL of 1X TBE in an Erlenmeyer flask
2. Heated in the microwave until fully dissolved (usually about 45 seconds to 1 minute)
a. Solution should be completely clear
b. Do in 30 second intervals
3. Allowed the solution to cool until comfortable to touch
4. Added 10 μL GelRed Nucleic Acid Gel Stain and mixed
5. Inserted casting tray, made sure the rubber on the sides is not overlapping
6. Carefully poured the agarose into the tray and placed the comb to create the wells
7. Allowed the gel to solidify
8. Once solidified, changed the orientation of casting tray where the rubber sides are not in contact with the sides of the system.
9. Poured in 1X TBE into the gel electrophoresis system to the fill line, being sure to submerge the gel, and removed the comb
Loading
1.Loaded ~5 μL of the ladder in the first well
2. Prepared your samples to load by adding in 1 μL of 5X Loading dye for every 5 μL of DNA and loaded
Running
1. Once the gel had been loaded, slid on the cover making sure the negative electrode is closest to the DNA and the positive electrode is at the bottom of the gel
2. Ran for about 45 minutes to an hour
a. Note: Longer with lower voltage gets you a better gel
Qiagen QIAEX II Gel Extraction Kit
1. Ran a restriction digest on the targeted DNA part using restriction enzymes and ran an agarose gel overnight
2. Cut the targeted DNA sequence out using a razor blade, made sure to get as much DNA while limiting the amount of agarose extracted
3. Pre-weighed an empty 50 mL falcon tube before adding the gel exicisions.
4. Added the gel extracts to the Eppendorf tubes and weighed again.
5. Calculated the mass of the gel using the difference of the two measurements.
6. Multiplied the mass by a factor of 3 to get the volume of Buffer QG needed.
7. Added the respective amounts of Buffer QG to each of the tubes
8. Vortexed the tubes until all the gel had dissolved
9. Added 1:1 ratio of Gel mass: Isopropanol
10. Once dissolved, added the solution into spin columns and centrifuged for 30 second at 13,000 rpm and carefully removed the supernatant. Note: One column can only handle .4 grams of agarose
11. Added 375 μL of Buffer QG to each of the spin columns, centrifuged for 1 minute, and discarded the flow through
12. Added 750 μL Buffer PE to each of the spin columns, centrifuged for 1 minute, and discarded the flow through
13. Centrifuged the spin columns empty to remove the residual buffer.
14. Added 50 μL Buffer EB to each of the spin columns and eluted the DNA into two different clean eppendorf tubes.
10X TBE
1. Dissolved 108 g Tris and 55 g Boric acid in 800 mL of diH2O
2. Added 40 mL 0.5 M Na2EDTA (pH 8.0)
3. Brought final volume up to 1 L
Terrific Broth media
1. 450 mL of deionized water was added to a 2L flask
2. 24 g of yeast extract , 20 g of tryptone, and 4mL of glycerol were added
3. The solution was stirred until the ingredients dissolved
4. The flask was covered with aluminum foil and autoclaved for 45 minutes
5. The solution was cooled
6. 100 mL of phosphate buffer was added and it was stored in the fridge
LB media
1. 10 grams Tryptone
2. 10 grams NaCl
3. 5 grams Yeast Extract
4. Filled up to 1 L with di wat
5. Autoclaved for 45 minutes
YM media
1. Dissolved the following in 900 mL of diH2O and bring up to 1 L
a. 0.4 g Yeast extract
b. 10 g Mannitol
c. 0.1 g NaCl
d. 0.2 g MgSO4ï 7H2O
e. 0.38 g K2HPO4
2. Autoclaved for 45 minutes and bring to a pH of 7
Purpose: To add a gene into a plasmid
1. Added 6 μL of diH2O to a clean 1.5 mL Eppendorf tube, 1μL of T4 DNA Ligase Buffer, 1μL of plasmid DNA and 1μL of DNA part and mixed.
2. Added 1μL of T4 DNA Ligase.
3. Pipet mixed the tube and incubated at room temperature for 10 minutes.
QIAprep Spin Miniprep Kit Protocol
1. Centrifuged 3 mL of bacterial overnight culture in two separate Eppendorf tubes (1.5 mL in each) at 8,000 rpm for 3 minutes at room temperature.
2. Discarded the supernatant and resuspended pelleted bacterial cells in one tube with 250 μL Buffer P1 and transferred to the other and resuspended until one eppendorf tube contained the pelleted cells resuspended in 250 μL Buffer P1.
3. Added 250 μL of Buffer P2 and inverted 5 times.
4. Added 350 μL of Buffer N3 and immediately mixed by inverting 5 times.
5. Centrifuged for 10 minutes at 13,000 rpm.
6. Micropipette 800 μL of the clear supernatant into a spin column and centrifuged for 60 seconds and discarded the excess liquid.
7. Add 500 μL of PB and centrifuged the spin columns for 60 seconds. Discarded the flow through.
8. Added 750 μL of PE to the spin columns, centrifuged for 60 seconds, and discarded the flow through.
9. Centrifuged the spin columns again for 60 seconds to remove residual wash buffer and discarded the flow through.
10. Transferred the spin columns to a clean eppendorf tube and added 50 μL of EB to the center of the spin column to elute the DNA.
11. Allowed the spin column to stand for one minute and then centrifuged for one minute.
12. Recorded the concentrations for each sample./p>
Purpose: To grow bacteria containing the introduced plasmid
To Thaw:
1. Antibiotics (Thaw @ room temp.) … Found in freezer
1. Added about 5-7 mL of LB to a 25 mL Falcon tube along with 5-7 μL of antibiotics (Chloramphenicol for pSB1C3; Kanamycin for pCB302; Ampicillin for DinoIII)
2. Dipped a p10 tip into your selected colony and dropped into the tube
a. If using a glycerol stock, simply scrapped some of the ice with the p10 tip and dropped into the tube
i. NOTE: did NOT let the glycerol stock defrost!
b. If you want to do overnight colony PCR from the same colonies:
i. Used the tip of a pipette or toothpick to pick a colony then swirled it around in 10 μL of DI water
ii. Added 1 μL into PCR tube and another μL into overnight cultures
3. Incubated in the water bath at 37° C at 220 rpm for 16-18 hours
Purpose: To save your cells with the introduced plasmid
Glycerol Stocks
1. Took 1 mL of 50% glycerol and 1 mL of the overnight culture (after incubation) and added to a glycerol stock tube.
2. Labelled with your name, date, and the contents and stored in the -80° C freezer in CLSO 442
Purpose: To amplify a sequence of DNA
To thaw:
1. Mastermix (on ice) … Found in freezer
2. Primers (on ice) … Found in freezer
20 μL Reaction
1. Prepared a PCR concentration cocktail with the following proportions: 7 μL of diH2O, 10 μL PCR Mastermix, 1 μL of the forward primer, and 1 μL of the reverse primer.
2. Added 19 μL of the concentration cocktail into a PCR tube along with 1 μL of the DNA.
2. Placed PCR tube in the thermocycler at the following generic settings:
a. 95° C f or 3:00 minutes
b. 95° C for 1:00 minute
c. 52° C for 1:00 minute *Annealing temperature varies depending on primer
d. 72° C for 1:00 minute
e. 30X (Go to Step 2)
f. 72° C for 5:00 minutes
Lid Temperature 105° C
Colony PCR Protocol
1. Prepared a PCR concentration cocktail with the following proportions: 7 μL of diH2O, 10 μL PCR Mastermix, 1 μL of the forward primer, and 1 μL of the reverse primer.
2. Added 19 μL of the concentration cocktail into a PCR tube.
3. Using a 10 μL micropipette, touched the tip onto the selected colony and swirled around in the PCR tube.
4. Placed PCR tube in the thermocycler at the following generic settings:
a. 95° C for 3:00 minutes
b. 95° C for 1:00 minute
c. 52° C for 1:00 minute *Annealing temperature varies depending on primer
d. 72° C for 1:00 minute
e. 30X (Go to Step 2)
f. 72° C for 5:00 minutes
Lid Temperature 105° C
Purpose: to cut at restriction sites and remove inserted DNA from plasmid.
To thaw:
1. Restriction enzyme (on ice) …. Found in freezer
2. Fast Digest buffer (on ice) … Found in freezer
3. Turn on water bath
30 μL Fast Digest Restriction Digest
1. Prepared a Fast Digest concentration cocktail with the following proportions: 1 μL Restriction Enzyme #1, 1 μL Restriction Enzyme #2, 3 μL of 10X Fast Digest Buffer, and 15 μL of diH2O.
2. Added 20 μL of this cocktail to a clean 1.5 Eppendorf tube and then added 10 μL of DNA
3. Incubated at 37° C for 30 minutes.
Restriction digest protocol
Purpose: To digest/linearize large amounts of plasmid for gel extraction
250 μL Fast Digest Restriction Digest
4. Prepared a Fast Digest concentration cocktail with the following proportions: 25 μL Restriction Enzyme #1, 25 μL of 10X Fast Digest Buffer, and 100 μL of diH2O.
5. Added 20 μL of this cocktail to a clean 1.5 Eppendorf tube and then added 100 μL of DNA
6. Incubated at 37° C for 3 hours.
Heat Kill
Purpose: To inactivate the restriction enzymes before a ligation is performed
1. Placed digested DNA in water bath at 65° C for 20 minutes