Team:MITADTBIO Pune/Experiments
"Try and try until your PCR starts working" ~ your average iGEMmer
Standardizing protocols was a crazy and time consuming journey for us. We therefore present to you firstly with a list of all the approaches we tried for transformation and making compentent cells, protocols for the procedures that did work for us, and assays we carried out for our project.
APPROACHES
Competent cells
| METHOD 1 | METHOD 2 | METHOD 3 | METHOD 4 |
|---|---|---|---|
| 1] 1ml of O/N DH5a culture in fresh 50 ml LB for 3 hours. Keep on ice for 30 min | 1]1ml of O/N DH5a culture in fresh 50 ml SOB for 3 hours | JM109 Competent Cells from Promega | 1]1ml of O/N DH5a culture in fresh 50 ml LB for 3 hours |
| 2]Centrifuge 25 ml for 10 min at 4000rpm | 2]Centrifuge 25 ml for 10 min at 3000rpm | 2]Centrifuge 25 ml for 10 min at 6000rpm | |
| 3]Add 200ul MgCl2 of 100 mM to pellet. Ice for 30 min | 3]Re-suspend in 6ml of CCMB80( tip : add 3ml first to resuspend the cells. When cells are in suspension, add another 3ml CCMB80 buffer for a total of 6ml ). Ice for 20 min | 3]Resuspend in 6.4ml of HTB | |
| 4]Centrifuged for 10 min at 4000rpm | 4]Centrifuged for 10 min at 3000rpm | 4]Centrifuged for 15 minutes at 3500rpm | |
| 5]Add 100ul CaCl2 of 100 mM to pellet. Ice for 20 min | 5]Re-suspend in 4ml of CCMB80 (tip : add 2ml first to resuspend the cells. When cells are in suspension, add another 2ml CCMB80 buffer for a total of 4ml) . Ice for 20 min | 5]Re-suspend in 1.6 ml of HTB. Add 120uL DMSO | |
| 6]Make aliquots of 50µl | 6]Make aliqouts of 50 ul. | 6]Make aliqouts of 200ul. |
Preparation of medium
1]SOC Broth/agar
| Mix | Quantity(100ml) |
|---|---|
| 0.5%yeast extract | 0.5g |
| 2% tryptone | 2g |
| 10mM NaCl | 0.2ml |
| 2.5mM KCl | 0.5ml |
| 20mM MgSO4 | 2ml |
| 10mM MgCl2 | 2ml |
| 20mM glucose | 2ml |
| Agar | 1.5g |
2]SOB Broth/agar
| Mix | Quantity(100ml) |
|---|---|
| 0.5%yeast extract | 0.5g |
| 2% tryptone | 2g |
| 10mM NaCl | 0.2ml |
| 2.5mM KCl | 0.5ml |
| 20mM MgSO4 | 4ml |
| Agar | 1.5g |
3]Luria Broth/agar
| Mix | Quantity(100ml) |
|---|---|
| Luria Broth | 2.0g |
| Luria agar | 3.5g |
4]CCMB80 buffer (pH 6.4)
| Mix | Quantity(100ml) |
|---|---|
| KOAc | 10 mM |
| CaCl2.2H2O | 80 mM |
| MnCl2.4H2O | 20 mM |
| MgCl2.6H2O | 10 mM |
| glycerol | 10% |
5]HEPES buffer(pH 7.4)
| Mix | Quantity(100ml) |
|---|---|
| 10mM HEPES | 2.38g |
Transformation
| METHOD 1 | METHOD 2 | METHOD 3 | METHOD 4 |
|---|---|---|---|
| 1] Add 1ul of Ligated product in 50ul of competent cells | 1]Add 10ul of Ligated product in 50ul of competent cells | 1]Thaw JM109 on ice . Add 1ul DNA . Mix well . | 1]Dilute plamid 1:10. Add 10ul in 200ul. |
| 2] Keep on ice for 30 min | 2] Keep on ice for 30 min | 2]Keep on ice for 30 min | 2]Keep on ice for 30 min |
| 3]Heat shock for 45 sec at 42˚ C | 3]Heat shock for 45 sec at 42˚ C | 3]Heat shock for 30 sec at 42˚ C | 3]Heat shock for 2 minute at 42˚ C |
| 4]Ice for 5 min | 4]Ice for 5 min | 4]Ice for 2 minutes | 4]Ice for 1 minute |
| 5]Add 950 ul of SOC media. Incubate for 1 hour. | 5]Add 900 ul of LB media. Incubate for 1 hour. | 5]Add 400 ul of SOC . Incubate 1 hour. | 5]Add 400 ul of SOB . Incubate for 1 hour. |
| 6]Spread plate and incubate. | 6]Spread plate and incubate. | 6]Spread plate and incubate. | 6]Spread plate and incubate. |
PROTOCOLS
Gradient PCR
| Component | Amound for 25μl Reaction |
|---|---|
| 5X Flexi buffer | 5 μl |
| dNTPs | 0.5 μl |
| Forward Primer | 1 μl |
| Reverse Primer | 1 μl |
| Template DNA | 1 μL |
| Taq DNA Polymerase | 0.2 μl |
| Nuclease free water | To 50 μL |
| MgCl2 | 0.2 μl |
| Step | Temperature | Time | Cycles |
|---|---|---|---|
| Initial Denaturation | 98°C | 5 min | 1 |
| Denaturation Annealing Extension |
95°C 64°C to 72°C 72°C |
20 sec 30 sec 30 sec |
30 |
| Final Extension | 72°C | 10 min | 1 |
Gel Electrophoresis
1. 1% Agarose gel (1gm in 100ml 1XTAE buffer) was melted.
2. EtBr was added and allowed to solidify.
3. 2ul loading dye with 10ul sample was loaded on gel.
4.Gel electrophoresis at carried out at 70V.
5. After loading dye reaches 70% of the length of the gel observe using gel doc.
Purification(Promega PCR cleanup kit)
1. Add the remaining PCR product to a column mounted on a collection tube.
2. Spin at 10,000 rpm for 1 min.
3. Discard the contents in the collection tube.
4. Add 700µl of membrane wash solution to the column.
5. Spin at 16,000rpm for 1 min.
6. Discard the contents in the collection tube.
7. Add 500µl of membrane wash solution to the column.
8. Spin at 16,000rpm for 1 min.
9. Discard the contents in the collection tube.
10. Spin at 16,000rpm for 1 min to remove excess ethanol.
12. Add 20µl of nuclease free water.
13. Spin at 16,000rpm for 1 min.
14. Add 20µl of nuclease free water.
15. Spin 16,000rpm for 1 min.
16. Store 40µl of purified product in -20˚C.
Ligation
Concentration of DNA =OD(@260nm)*50*50
Ligation=(ratio*kb of insert*100)/(kb of vector)
Component
Amount for a 10 μl Reaction
T4 DNA Ligase Buffer with ATP (10x)
1 μl
Vector Backbone
25-50ng
Insert DNA
25-100ng
(3:1 molar ratio of insert:vector)
T4 DNA Ligase
0.5 μl
ddH2O
To 10 μl
| Component | Amount for a 10 μl Reaction |
|---|---|
| T4 DNA Ligase Buffer with ATP (10x) | 1 μl |
| Vector Backbone | 25-50ng |
| Insert DNA | 25-100ng (3:1 molar ratio of insert:vector) |
| T4 DNA Ligase | 0.5 μl |
| ddH2O | To 10 μl |
Competent cells
1. Inoculate JM109 in LB medium. Let it grow overnight.
2. Inoculate 1ml of overnight grown culture into fresh LB medium of 25ml.
3. Incubate for 3 hours. Check OD should be around 0.3.
4. Transfer this culture into centrifuge tubes of 2ml .
5. Spin at 15,000 rpm for 5 min at 4˚C.
6. Gently re-suspend in 500 μl of CaCl2 (10mM) and incubate in ice for 30 min.
7. Spin at 15,000rpm for 5 min at 4˚C.
9. Discard the supernatant and resuspend in 50 μl of CaCl2.
11. Incubate in ice for 2 hrs.
Transformation
1. Add 1µl of ligated product in 50µl of competent cells.
2. Incubate on ice for 45 min.
3. Heat shock for 45sec on 42˚C.
4. Incubate in ice for 5 min.
5. Add 1 mL LB and incubate at 37˚C for 1.5 hour at 120-200rpm.
6. Centrifuge tube at 5,000rpm for 7 mins.
7. Resuspend the pellet in 100 μl supernatant at spread plate at chloramphenicol plates.
Colony PCR
| PCR mix | Amound for 25μl Reaction |
|---|---|
| 5X Flexi buffer | 10 μl |
| dNTPs | 0.5 μl |
| Forward Primer | 1 μl |
| Reverse Primer | 1 μl |
| plasmid | colony |
| Taq DNA Polymerase | 0.4 μl |
| Nuclease free water | To 25 μL |
| MgCl2 | 2 μl |
| Step | Temperature | Time | Cycles |
|---|---|---|---|
| Initial Denaturation | 95°C | 5 min | 1 |
| Denaturation Annealing Extension |
95°C 54°C 72°C |
1 min 1.5 min 1min |
30 |
| Final Extension | 72°C | 10 min | 1 |
Alkaline lysis
1. Grow the transformants in 1 ml LB with appropriate antibiotic.
2. Take the culture and spin at 5,000rpm for 3 mins at 4˚C.
3. Re-suspend cell pellet in 50µl chilled solution I.
4. Incubate in ice for 5 min.
5. Add 100µl of solution II. Mix gently.
6. Incubate in ice for 10 min.
7. Add 75µl of solution III. Mix gently.
8. Incubate in ice for 15 min.
10. Centrifuge at 12,000rpm for 15 min at 4˚C.
Solution I (pH 8):
1. 50mM glucose
2. 10mM EDTA
3. 25mM Tris-HCl
Solution II :
1. 0.2% NaOH
2. 1% SDS
Solution III:
1. 3M Potassium / sodium acetate
Plasmid Cleanup
1. After alkaline lysis for plasmid isolation centrifuge at 12,000rp,for 15 min at 4˚C.
2. Treat the supernatant with 20µl of Rnase A.
3. Incubate at 37˚C for 30 min at 200 rpm.
4. Equal volume of phenol : chloroform : isoamyl alchohol was added.
5. Keep in ice for 10 min.
6. Spin at 10,000rpm for 10 min at 4˚C.
7. Collect the aqueous phase.
8. Add 150mM NaCl. Mix well.
10. Add 2ml of absolute chilled ethanol. Mix gently.
11. Incubate for 1 hour in -20˚C.
12. Spin at 10,000rpm for 15 min at 4˚C.
13. Wash with 70% ethanol.
14. Spin at 10,000rpm for 10 min at 4˚C.
15. Discard the supernatant.
16. Air dry.
17. Re-suspend in 20µl of autoclaved water.
SDS Page
Reagents :
~30% Acrylamide (pH 7):
1. Add 290g acrylamide.
2. 10g of N,N'-methylbisacrylamide in 600ml of distilled water.
3.Make up the volume to 1L with distilled water after adujsting the ph.
(tip:Heating may be necessary to dissolve the acrylamide.)
~Bisacrylamide:
1.Add 29.22 g of acrylamide.
2.0.78 g of bisacrylamide.
3.Add 100 ml of distilled water.
~1.5M Tris-HCL(ph8.8):
1.Add 90.75g of Tris-free base.
2.Add 300ml water.
3.Add 8ml of concentrated HCL.
4.Adjust pH to 8.8 .
5.Make the volume upto 500ml.
~1M Tris-HCL (pH 6.8):
1.Add 60.54g of Tris-free base.
2.Add 300ml of water.
3.Add 36ml of concentrated HCL.
4.Adjust the pH to 6.8.
5.Make up the volume to 500ml.
1. Resolving gel :
| Components | Amount |
|---|---|
| ddH2O | 1 mL |
| 1.5 M Tris/HCl (pH 8.8) | 3.0 mL |
| 30% A/C stock | 3.2 mL |
| 10% w/v SDS | 120 µl |
| 10% w/v APS | 120 µl |
| TEMED | T5 µl |
2. Stacking gel:
| Components | Amount |
|---|---|
| ddH2O | 3.6 mL |
| 1.5 M Tris/HCl (pH 6.8) | 1.56 mL |
| 30% A/C stock | 0.6 ml |
| 10% w/v SDS | 20 µl |
| 10% w/v APS | 140 µl |
| TEMED | 5 µl |
3. Butanol : 50 mL ddH2O + 30mL butanol
4. 2X running buffer : For 8mL
| Components | Amount |
|---|---|
| ddH2O | 2.8mL |
| 0.5M Tris/HCl (pH 6.8) | 1 mL |
| glycerol | 0.8 ml |
| 10% w/v SDS | 3.2mL |
| 0.2% bromophenol blue | 0.2mL |
5. 5X running buffer : For 8mL
| Components | Amount |
|---|---|
| Tris-HCl pH 8.3 | 125mM |
| Glycine | 1.23M |
| SDS | 0.5% |
6. Staining and destaining solution:
Coommassie G250= 0.5%
Ethanol = 30%
Acetic acid= 10%
Procedure :
1.Assembling the glass plate
Assemble the glass plate on a clean surface. Lay the longer glass plate (the one with spacer) down first, then place the shorter glass plate on top of it. Embed them into the casting frame and clamp them properly make sure that the bottom ends of the glass plates are properly aligned. If so place it on the casting stand.
2. Casting the gel
Prepare 12% Resolving gel and 5% stacking gel using the composition given above. Add APS and TEMED to the monomer solution (just before pouring) and mix well by swirling gently. Pour the solution till the mark. Add a layer of water saturated butanol or distilled water on top of the gel so as to level the poured gel). Allow the gel to polymerize for 20-30 minutes. Prepare the stacking gel monomer solution. Combine all reagents except APS and TEMED. Drain the water saturated butanol with strips of filter paper. Add APS and TEMED to the monomer solution (just before pouring) and mix well by swirling gently.(Make sure you keep the comb ready by the side). Immediately place a comb in the stacking gel sandwich. Allow it to polymerize for at least 20 minutes.
3.Preparation of samples
Samples were centrifuged at 6000 rpm for 10 min. Samples were then mixed with an equal volume of 2 x non reducing samples buffer. Boil for 10 mins, centrifuge at 5000 rpm for 5 min and 15 - 20 μl loaded per well. 10ul of denatured molecular weight markers were also electrophoresed on each gel.
4.Running the gel
To assemble, take out the gels from the casting frame and clamp them in the gel apparatus (Make sure that the short plate always faces inside). When the plates are secured, place them in tank. Fill the inner chamber of the tank with buffer. Remove the comb carefully. Insert the loading tip to a few mm from the well bottom and deliver the samples into the well. Rinse the syringe a few times with distille water after loading. Attach the power supply by putting the lid. Set the voltage up to 150 V and run for 1 hour till the dye front reaches the end of the gel.
5. Gel washing
Take out the gel and wash (incubate 1 hr at room temp on a rotating shaker to remove the SDS and renature your proteinases) with 2.5% TritonX100 in 50 mM tris pH 7.4, 5 mM CaCl2, 1 μM ZnCl2. Rinse briefly with deionized water. Incubate overnight at 37 °C with 50 mM Tris pH 7.4, 5 mM CaCl, 1 μM ZnCl2.
6.Staining and Destaining
Stain with 0.5% Coomassie G250 for 30 minutes (entire gel should be dark blue) De-stain in 30% Ethanol/10% acetic acid until you see clear bands (a couple of minutes). Change to 2% acetic acid to stop staining. Rehydrate in 2% acetic acid overnight
Restriction Digestion
1. RE mix for plasmid digestion :
| Components | Amount (50 µl) |
|---|---|
| Nuclease free water | 41 µl |
| Buffer (10X) | 5 µl |
| BSA (100X) | 1 µl |
| Plasmid | 2 µl |
| RE | 1 µl |
2. RE mix for DNA insert digestion :
| Components | Amount (50 µl) |
|---|---|
| Nuclease free water | 40 µl |
| Buffer (10X) | 5 µl |
| BSA (100X) | 1 µl |
| DNA insert | 3 µl |
| RE | 1 µl |
2. Incubate at 37˚C in hot water bath for 1 hour.
RE cleanup (Using Promega cleanup kit) :
1. Binding solution (50 µl) + RE-DNA/plasmid mix (50 µl)
2. Put the mix in the column. Incubate for 1 min.
3. Centrifuge at 16,000 rpm for 1min.
4. Decant. Add 700µl of wash buffer.
5. Centrifuge at 16,000 rpm for 1min.
6. Decant. Add 500µl of wash buffer.
7. Centrifuge at 16,000 rpm for 1min.
8. Add the column in a new tube. Add 15 µl of nuclease free water in the column and incubate for 5 mins.
9. Centrifuge at 16,000 rpm for 1min. The supernatant has cleaned DNA.
Fluorescence microscopy
1. Incubate the primary culture of potassium promoter with GFP and GFP with no promoter for 18 h at 37˚C in 2ml of Luria Broth.
2. Add different concentration of potassium in Luria Broth.
3. To this add 25ul of primary culture .
4. Incubate overnight for growth.
5. Make a glass slide with this culture.
6. Cover it with a cover slip.
7. Observe under fluorescent microscope.
ASSAYS
Blood collection and analysing serum
Blood was collected in heparin tubes to prevent clotting , to collect equal amount of serum from 2 ml aliquots. Blood was centrifuged to collect serum to be used for analyzing the electrolyte present in it. This analyzing was done using electrolyte analyzer in Vishawaraj Hospital ,Loni. This is an important aspect of our project as we will be dealing with blood in the sanitary napkins. This provides us with the concentration of potassium which is required to induce our promoter in a particular range. Blood was also used to check the interaction with Laccase enzyme whether it or it does not effect the functionality.
Fluorescent measurement
This was also performed in presence of serum to check if there were any hinderance with the components of blood.
1.Inoculate primary culture in LB.
2.Grow it over night.
3.Take OD of the sample.
4.Calculate the culture to be taken to dilute it to 0.1 OD.
5.Wash three times with 0.8% NaCl.
6.Resuspend pellet in 2ml K0 media.
7.Take 25µl of suspended cells and inoculate in 2ml media of different concentration of KCl.
8.Incubate overnight and one more set for 2-3 hours.
Reagent :
K0 minimal media
| Reagents | Working concentration |
|---|---|
| Na2HPO4 | 46 mM |
| NaH2PO4 | 23 mM |
| (NH4)2SO4 | 8 mM |
| MgSO4 | 0.4 mM | Citric acid | 0.66 mM |
| Tri-sodium citrate dehydrate | 0.33 mM |
| Thiamine hydrochloride | 1 mg/liter |
| Glucose | 2 g/liter. |
| Casein acid hydrolysate | 0.2% |
Laccase assay
Laccase enzyme are blue copper oxidases having versatile substrate like phenolics, aromatic amines, and other electron-rich substrates by reducing O2 to H2O.
Laccase activity was determined by the oxidation of ABTS method. ABTS is oxidized by laccase to cation radical. The concentration of this radical is responsible for the intense blue-green color that shows enzyme activity which is to be read at 420 nm in a spectrophotometer.
This assay was performed in presence of blood as well to check if Laccase is induced in presence of blood due to iron , as laccae being a metalo-enzyme ; co factor as copper .
1]Make a mixture of 200ul of 0.5 mM ABTS,1.5ml 0.1 M sodium acetate (pH 4.5), add 100 μL of culture and 200ul of supernatant
2]Incubate for 5 min.
3]Absorbance was read at 420 nm in a spectrophotometer against a suitable blank.
Crystal violet assay
1]Biofilms were grown by subculturing 18 h cultures to a starting OD600 of 0.2 and immediately diluting them 1:100 in BHI supplemented with 0.4% glucose.
2]A 5 ml aliquot of each strain was added to each of the 3 wells.
3]Biofilms were allowed to shake at 100 rpm at 37°C for 18 h. To harvest the biofilms, the media was aspirated off and the biomass was washed twice with 5 ml of sterile water.
4] The biomass was allowed to air dry and stained for quantification.
5]To assess biomass, biofilms were stained with 500 μl of 0.1% crystal violet for 10 min at room temperature.
6]Excess dye was removed and biofilms were washed with 5 ml of sterile water.
7]Add 200ul acetic acid and read absorbance at 580nm.
Cover slip assay
1]Biofilms were grown by subculturing 18 h cultures to a starting OD600 of 0.2 .
2]These subcultures were then incubated with a cover slip in with luria broth and crystal violet.
3]These slips were washed .
4] Visualization was observed.