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Team:ATOMS-Turkiye/Protocol2
From 2014.igem.org
Protocols
1-) Wash the cells in the flask with 5 mL 1X PBS twice. At this stage removing PBS completely is very important. Otherwise activity of Trypsin decreases
2-) Add 2 mL Trypsin on cells. Be careful; Trypsin should be spreaded whole surface. Then wait 5 minute at 37°C. Because trypsin works maximum activity at 37 ° C.
3-) After we sure that cells remove from surface completely, wash the surface carefully with 5 mL medium via pipetting .
4-) Take all the mixture which we prepared to falcon tubes( 15 mL) and centrifuge 6 minutes.
5-) Supernatant should be taken carefully without damaging to cells.
6-) Add 5 mL medium on pellet and dissolve the pellet in medium.
7-) Take 100 mL from cell mixture, take 100 mL from Tripan-Blue and mix in eppendorf.
8-) Take 20 micro liters to Hemocytometer( Thoma lam) and count after then.
9-) We will put the lam to microscope. Lam’s H zone should be under the light.
10-) There are 5 areas that should be counted.
11-) Total number of cells which at 5 areas, are counted at arithmetic average is counted.
12-) To count the total cell number;
*Cell number: Average cell count x dilution factor x
*Dilution Factor: It is the rate between dye and cell contents. For this protocol dilution factor is 2.(1+1)
*For example; 68x2x=1.360.000(cell count per mL). But we want to seed 500.000 cell per mL.
1 mL 1.3 million
X 0.5 million
X= 0,385 mL = 385 microliters
*So we will take 0.385 microliters to seed 500.000 cells.
*According to the material ( where will we seed our cells? Flask, petri, well..) amount of medium is counted and the cells are seeded.
*For example, according to six well-plate; 2 mL medium is put, Than 385 microlitres cells are added.
*For six well-plate, the best situation is adding 500.000 cells.
1 -) The medium stored at +4 ° C, is heated at 37 ° C at least 30 minutes.
2 -) Remove all mediums from the flasks carefully without damaging the cells. At this stage, the medium is in the flask, can be poured directly or can be taken with pipette.
3 -) 10 mL medium is taken which heated and clean, is added to in the middle of flask. This stage is done very slowly and absolutely avoiding the cells. After than flask is careened slowly (medium should not touch to flask’s cap) to mix the cells with medium.
4-) While changing medium, we must be careful and fast as much as we can.
*The amount of solution according to flask type;
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1 -) Set Benmari at 37 ° C.
2 -) The cell line stored at -80 ° C, is brought to cell culture room in ice. Then put in 37 °C and wait 2 minutes.
3 -) After melting cells for 2 minutes, cryovial tubes are cleaned with alcohol and their caps are opened.
4 -) All the stock cells are put in to 5 mL medium placed 15 mL falcon tubes.
5 -)Do pipetting the Medium-Cell mixture carefully. At this stage, it is important not to harm the cells. So don’t work fast, don’t hurry. Work slowly and gentle.
6 -) Put 9 mL medium is to 75 mL empty flask.
7 -) Add the mixture, which was prepared in falcon tube, to flask. Shake gently to the left and the right way up and down carefully. Take care to never touch the mixture to flask’s cap.
8 -) Put the cells into the incubator and set at 37 ° C and %5 CO2.
The amounts of solution according to flask type
- Pick a single colony into 5ul of NFW. (Fresh colonies grown that day work best, but they can also come from 4 C).
- Boil for 5 min at 95C.
PCR Reaction
Keep all the reagents at 4C while preparing the mixture. Pre-heat the thermocycler to 95C and transfer your reaction directly from 4 C.
For E.coli(DH-5α)
1-) Streak DH5α directly from a frozen stock onto LB agar plate (Amp-).
2-) Incubate at 37℃ for 12 h.
3-) Inoculate one well isolated colony into 5ml of LB(Amp-).
4-) Incubate at 37℃ until OD600 =0.8
5-) Transfer the preculture 5ml to 250ml of LB(Amp-)
6-) Incubate at 23℃ until OD600 =0.4-0.5 (120rpm/min )
7-) Place sample on ice for 10min
8-) Transfer the culture to steril ice cold tube(250ml)
9-) 3.5Krpm at 4℃ for 5min
10-) Remove the sup well
11-) Resuspend the pellet by gently vortexing and pippeting in 100 ml of ice cold TB*
12-) Sit on ice for 10min
13-) 3K rpm at 4℃for 5min
14-) Remove the sup well
15-) Resuspend the pellet gently in 25ml of TB
16-) Add 875μl DMSO with disposable pippet
17-) Mix gently by swirling
18-) Sit on ice for 10min
19-) Add 875μl DMSO
20-) Mix gently by swirling
21-) Sit on ice for a few minuites
22-) Dispense the sample into 1.5ml tube sterilized with UV for 10min
23-) Chilled in liquid N2
[Preparation TB]
1-) Add the following into 475 ml milli-Q
2-) PIPES 1.5g
3-) CaCl2･2H2O 1.1g
4-) KCl 9.3g
5-) Adjust pH to 6.7 with 1N KOH
6-) Add 5.45g of MnCl2･4H2O
7-) Add milli-Q upto 500ml
8-) Filtration
Standard %0.8 Agarose Gel Preparation
- Measure out 0.8 g of agarose
- Pour agarose powder into a microwavable flask along with 100mL of 1xTBE
- Microwave for 3 mins (until the agarose has dissolved completely and there is a nice rolling boil).
- Let agarose solution cool down for 5min.
- Add 3.6 ul EtBr and pour the agarose into a gel tray with the suitable well comb in place (pour slowly to avoid bubbles which will disrupt the gel).
Wait 20-40 mins until poured gel has completely solidified.
Gel Purification
Gel purification using&Thermo Scientific GeneJET Gel Extraction Kit
-All purification steps should be carried out at room temperature.
-All centrifugations should be carried out in a table-top microcentrifuge at sup12000 x g
Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.
- Note. If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.
- Add 1:1 volume of Binding Buffer to the gel slice (volume: weight)(e.g., add 100 ul of Binding Buffer for every 100 mg of agarose gel).
Note. For gels with an agarose content greater than 2%, a dd 2:1 volumes of Binding Buffer to the gel slice. - Incubate the gel mixture at 50-60°C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
- Optional: use this step only when DNA fragment is inf 500 bp or sup10 kb long. If the DNA fragment is inf 500 bp, add a 1:2 volume of 100% isopropanol to the so lubilized gel solution (e.g. 100 ul of isopropanol should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly. If the DNA fragment is sup10 kb , add a 1:2 volume of water to the solubilized gel solution (e.g. 100 ul of water should be added to 100 mg gel slice solubilized in 100 ul of Binding Buffer). Mix thoroughly.
- Transfer up to 800 ul of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
Note. If the total volume exceeds 800 ul, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through aftereach spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1 g of total agarose gel per column. - Optional: use this additional binding step only if the purified DNA will be used for sequencing. Add 100 ul of Binding Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
- Add 700 ul of Wash Buffer (diluted with ethanol as described on p. 3) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
- Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.
Note. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions. - Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge tube (not included). Add 50 ul of NFW to the center of the purification column membrane. Centrifuge for 1 min.
Note. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 ul does not significantly reduce the DNA yield. However, elution volumes less than 10 ul are not recommended. If DNA fragment is sup10 kb, prewarm Elution Buffer to 65°C before applying to column. If the elution volume is 10 ul and DNA amount is inf5 ug, incubate column for 1 min at room temperature before centrifugation. - Discard the GeneJET purification column and store the purified DNA at -20°C.
Project Data
The data of our new favourite parts
- BBa_K1456005 Oxygen dependent degradation (ODD) domain from HIF-1α : ODD is a peptide sequence which is targeted by ubiquitin-proteosome degradation pathway when enough oxygen is present within the media as a result of the hydroxylation process. In hypoxic conditions, this process is inhibited and ODD can survive with its cohesive components. Therefore we aim to use this feature in designing a novel hypoxia sensing device.
- BBa_K1456006 Hypoxia Response Element (HRE) HREs are transcription factor binding sites for the hypoxia inducible factor-1alfa. In hypoxic conditions, the yield of HRE including promoter increases significantly. This part is planned to be used in the main sensor device of our design.
- BBa_K14569999 Kappa-B Response Element (kB-RE) kB-RE, a naturally sensing operon of hypoxia inducible factor 1alfa, is activated in some circumstances which pose cellular stress such as inflammation or oxidation. ROS presence also results in the transcription on kB-RE; therefore we intend to use it as ROS inducible promoter.
- BBa_K1456003 Superoxide dismutase-1 (SOD-1): SOD-1 is a natural and potent antioxidant enzyme existing in most of the human cells. It converts free radicals and reactive oxygen species, which are responsible for the oxidative damage, into hydrogen peroxide that is less harmful. It has three subtypes with various locations; therefore we have chosen SOD-1 for its optimum and ideal region in the cell.
- BBa_K1456002 Glutathione Peroxidase-1 (GPx-1) GPx-1 is another antioxidant enzyme which takes over the duty from SOD-1 by detoxifying hydrogen peroxide radical into water and oxygen. GPx-1 subtype of this enzyme has been decided to use in our project because of its convenience with our design.
- BBa_K1456001 Human Tissue Plasminogen Activator (tPA): tPA activates zymogen protein, plasminogen, which begin to cleave the main component of the clot, fibrin. It is the most potent drug being used clinically, so far. This part will help us to dissolve the hypoxia causing clot in a vessel.
- BBa_K1456003 Aprotinin: Oxygen Radical Inhibitor Aprotinin is a serine protease inhibitor which reacts with naturally occurring proteases present in the cell to prevent possible enzyme conversions. One of these interferences is the transformation of xanthine dehydrogenase, which can produce almost 80% of free radicals by becoming xanthine oxidase. Aprotinin can prevent the production such radicals by inhibiting this reaction.
We have designed and characterized the following parts:
