Team:IISER-Pune-India/Protocols

Mutatis Mutandis

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Protocols

Protocols Used in Laboratory

Preparation of Super Optimal Broth
For 1 L of SOB we use
5g Yeast Extract - 0.5%, 20g Tryptone - 2%, 0.584g NaCl - 10mM, 0.186 KCl - 2.5mM, 2.4g MgSO4.7H2O - 20mM

Mix dry ingredients and add 700mL dH2O
Add 1 NaOH dropwise to bring the pH of the solution to 7.5
Make up the volume to 1L
Autoclave
Note: Always wear gloves when using strong acids or bases.

Preparation of Super Optimal Agar
For 1 L of SOA we use
5g Yeast Extract - 0.5%, 20g Tryptone - 2%, 0.584g NaCl - 10mM, 0.186 KCl - 2.5mM, 2.4g MgSO4.7H2O - 20mM, 15g Agar - 1.5%

Mix dry ingredients (except agar) and add 700mL dH2O
Add 1 NaOH dropwise to bring the pH of the solution to 7.5
Add agarose to the solution
Make up the volume to 1L and mix thoroughly
Autoclave
Note: Always wear gloves when using strong acids or bases.

Preparation of Luria Bertini Broth

For 1 L of LB Broth, mix 25g LB Powder (10g Yeast Extract, 5g Tryptone, 10g NaCl) in dH2O
Autoclave

Preparation of Luria Bertini Agar

For 1 L of LB Broth, mix 25g LB Powder (10g Yeast Extract, 5g Tryptone, 10g NaCl) in dH2O
Mix 15g Agar thoroughly
Autoclave

Preparation of M9 Minimal Media
For 100mL of M9 Media
20mL of 5x M9 salt solution - 1x, For 100 mL M9 salts
Na2HPO4.7H2O - 6.4g, KH2PO4 - 1.5g, NaCl - .25g, NH4Cl - .5g, ddH2O - 100mL Autoclave
2mL of 20% Glucose solution 0.4% Filter Sterilized
200ul of 1M MgSO4 Autoclaved
10uLof 1M CaCl2 Autoclaved
78 mL of ddH2O Mix all solutions and do not autoclave.

Preparation of CCMB80 Buffer (For making competent E. coli)
For 1 L of the solution:
10mL of 1M KOAc solution - 10mM, 11.8g CaCl2.2H2O - 80mM, 4g MnCl2.4H2O - 20mM, 2g MgCl2.6H2O - 10mM, 100mL Glycerol - 10%
Mix all the salts in 700 mL ddH2O
Adjust the pH to 6.4 using 0.1N HCl solution
Add 7the Glycerol and make up the volume to 1L
Filter sterilize using 0.22 micron filter and syringe inside a laminar hood
Store at 4℃ in an autoclaved bottle

Preparation of 50x Tris-Acetate-EDTA Buffer (For use in Electrophoresis)
For 1 L of 50x TAE solution
242g Tris Base - 2M, 57.1mL Glacial Acetic Acid - 1M, 100mL EDTA Solution (pH 8.0)- 50mM
In 700mL ddH2O, mix Tris, Acetic Acid and EDTA Solution until dissolved
Make volume up to 1L and store at room temperature Note: Always wear gloves when using strong acids or bases. Pour Glacial Acetic Acid in a chemical fume hood only.

Preparation of 1x Tris-Acetate-EDTA Buffer (For use in Electrophoresis)
For 1 L of 1x TAE Buffer, mix 20 mL of 50x TAE buffer and make the volume up to 1L
Store at Room Temperature

Preparation of 500mM EDTA Solution

For 200mL of 500mM EDTA solution, mix 37.22g Disodium EDTA in 100mL dH2O
Bring the pH of the solution to 8.0 by adding NaOH pellets or adding 10N NaOH solution dropwise.
Make up the volume to 200mL
Autoclave Note: Adding NaOH to water is an exothermic reaction which will heat up the solution and increase the solubility of EDTA. Thus, pH should be carefully noted. Always wear gloves while handling strong acids or bases.

Preparation of 2M Tris Solution

For 100mL of 2M Tris solution, mix 24.2g Tris Base in 50mL dH2O
Bring the pH of the solution to 8.0 by using 10N HCl solution dropwise
Make up the volume to 100mL
Autoclave Note: Adding NaOH to water is an exothermic reaction which will heat up the solution and increase the solubility of EDTA. Thus, pH should be carefully noted. Always wear gloves while handling strong acids or bases.

Preparation of 1% Agarose Gel (For Electrophoresis)
For 50mL of gel
0.5g Agarose - 1%, 50mL 1xTAE Buffer, 2µL EtBr solution
Measure agarose and add it to TAE Buffer
Heat the flask in a microwave to dissolve the agar- Avoid bubbling; stop heating whenever bubbling starts and then heat
again for short intervals. The solution will become clear when ready
Ready the gel apparatus of the required size and ensure that it is leak-proof. Add the comb with required wells
Check the temperature of the flask. When it seems cool enough to touch, add the EtBr
Swirl gently, avoiding the formation of any bubbles, to mix the EtBr
Pour immediately into the apparatus and let cool Note: Use only dedicated flasks, gloves and tips for EtBr use. Discard tips, gloves, used gels, running buffer etc. in EtBr Waste only. Use insulated gloves/ hand protectors to handle hot apparatus.

Preparation of 1000x Chloramphenicol Solution
To make 5mL of 1000x Chloramphenicol

Measure 170mg of Chloramphenicol and add 5mL of 100% Absolute Ethanol
Mix well by tapping or vortexing
Using 0.22 micron filter and syringe, aliquot 1mL into 1.5mL tubes in a laminar hood
Store in -20℃ when not in use. Store in 4℃, if in use

Pouring LB Agar or SOA Plates (5 plates - 100mL)

Melt 100mL of agar solution such that no solid lumps remain
Let the solution cool till you can touch the flask with your wrist
Add 100µL of the 1000x Antibiotic Stock Solution
Gently swirl the agar solution and pour into open plates immediately, inside the laminar hood
Let the agar solidify
Store plates in 4℃ or use for plating immediately
Note: Use insulated gloves/ hand protectors to handle hot apparatus. Antibiotic may degrade if put in when the agar is too hot.

Preparation of Ultracompetent E. coli cells

From a glycerol stock or a culture plate, inoculate a single colony in 2mL SOB and culture overnight at 37℃ at 250rpm
Inoculate 100mL of SOB with 400µL of primary culture. Incubate at 20℃, 250rpmfor 19-22 hrs (until the OD600 of the secondary culture is 0.25~0.4)
Transfer to prechilled 50mL tubes and cool on ice
Centrifuge at 3000xg 4℃ for 10 min. Discard the supernatant
Resuspend the pellet in 3mL ice-cold CCMB80
Incubate on ice for 20 min
Centrifuge at 3000xg 4℃ for 10 min. Discard the supernatant
Dissolve pellet in 400µL ice-cold CCMB80
Measure OD600 of the cell suspension by diluting 50µL cells + 950µL SOB (For blank, substitute cells with 50µL CCMB80)
The OD600 of the suspension is 20x the OD600 of the measured sample. Dilute suspension by adding CCMB80 such that the final suspension has OD600 between 1-1.5
Aliquot 50µL/250µL in 1.5mL tubes
Incubate on ice for 20 min
Flash Freeze using liquid N2
Store at -80℃ in cryo box

Transformation of Competent E. coli cells

Spin down DNA sample at 9000xg for 2 min at 4℃
In a prechilled tube, add 1µL of DNA (Use ddH2O for control)
Add 50µL of the competent cells and mix by tapping gently
Incubate on ice for 30 min
Heat-shock in hot water bath at 42℃ for 60 sec
Incubate on ice for 5 min
Add 950µL of LB/SOB and mix gently
Incubate at 37℃, 250 rpm for 2 hours
Pellet the culture at 6000xg, 25℃ for 5 min
Discard the supernatant and resuspend the pellet in 100µL od LB/SOB
Spread the mixture on the required antibiotic plate
Incubate at 37℃ overnight (14-16 hrs)

Plasmid Isolation from bacterial cells
a. HiPurA™ Plasmid DNA Miniprep Purification Kit
The protocol is available online here: http://himedialabs.com/TD/MB508.pdf
b. Qiagen Plasmid Mini Kit

Take cells containing required plasmid and culture them overnight at 37℃
Take 3mL of the culture and centrifuge at 6000xg for 15min at 4℃
Discard the supernatant. Resuspend pellet in 0.3mL Resusupension Buffer P1
Add 0.3mL of Lysis Buffer P2. Invert mix 4-6 times and incubate for 5 min.
Add 0.3mL Neutralisation Buffer P3. Invert mix 4-6 times and incubate for 5 min on ice
Centrifuge the tube at 18000xg for 10 min at 4℃
Meanwhile, equilibrate the tip with 1 ml of QBT (Equilibration Buffer)
Carefully apply all of the supernatant to the tip
Wash the column twice with 2 mL of QC (Wash Buffer).
Place a collection tube and elute DNA by passing 0.8mL of QF Buffer
Add 0.56mL Isopropanol and mix. Centrifuge at 15000xg for 30 min at 4℃
Decant supernatant carefully. Wash pellet with 1mL 70% ethanol. Centrifuge at 15000xg for 10 min at 4℃
Decant supernatant. Air dry the tube for 5-10 min
Redissolve in 20µL QF Elution Buffer
c. Alkaline Lysis Prepare stock of Solution 1 and Solution 3 :- Solution 1:- 25mM of Tris.HCl (pH=8), 10mM of EDTA (pH=8), 50mM Glucose
Solution 3 :- 5M Potassium acetate - 60ml, Glacial acetic acid - 11.5ml, H2O - 28.5ml, Prepare fresh Solution 2:- 0.2N NaOH + 1% SDS

Now, take a 5ml overnight culture and harvest the cells by centrifuging the culture @6000rpm, 25°C for 10mins. Discard the supernatant.
Add 100μl of Solution 1 and resuspend the pellet. The solution will be opaque.
Add 200μl of Solution 2 and mix well. Do not shake vigorously. The solution will turn a clear.
Add 150μl of Solution 3 and mix well. Do not shake vigorously.
Incubate in ice for 5-10mins.
Centrifuge @12000rpm, 25°C for 10mins. There will be a white pellet with clear supernatant. Take the supernatant carefully into a fresh 1.5ml tubei. If the solution is not clear centrifuge again for 1min and take the clear solution in a fresh tube.
Add 150μl of isopropanol. Mix it well by inverting the tube a few times.
Centrifuge @13000rpm, 25°C for 10mins. Discard the supernatant.
Add 300μl of 70% ethanol and mix it well.
Centrifuge @13000rpm, 25°C for 10mins. Discard the supernatant.
Leave it for air-drying for 20mins. Make sure it is dry. The pellet turns translucent.
Dissolve the pellet in 30μl of Elution Buffer (EB).
Add 2μl of RNAase and incubate at 37°C for 3hrs.
Now, add 750μl of Loading Buffer (PB) and leave it for 5mins.
Load the solution into a Qiagen mini-prep column. Centrifuge @13000rpm, 25°C for 1min. Discard the flowthrough.
Add 210μl of Wash Buffer (PE) and centrifuge @13000rpm, 25°C for 2mins. Discard the flowthrough. Centrifuge again for 2mins.
Move the column to a fresh 1.5ml tube. Add 50μl EB and centrifuge @13000rpm, 25°C for 2mins.
Remove the column and proceed for measuring Nanodrop values for the concentration of plasmid DNA obtained.
Store the DNA at -20°C.

Polymerase Chain reaction (PCR)
a. For single part amplification
1. Using PrimeSTAR GXL polymerase
Make reaction mix containing:1) 10-30 ng of template, 2) 10 uL GXL buffer (1x stock) , 3) 4 uL dNTP mix (200 mM each), 4) 1 uL PrimeSTAR GXL polymerase, 5) 1 uL Forward primer (0.3 uM) , 6) 1uL Reverse primer (0.3 uM), 7) NFW to 50 uL
2.Using Pfu DNA polymerase
Make reaction mix containing:1) 10-30 ng of template, 2) 5 uL Pfu buffer (1x) , 3) 4 uL dNTP mix (200 mM each), 4) 2 uL Pfu polymerase, 5) 1 uL Forward primer (0.3 uM), 6) 1uL Reverse primer (0.3 uM), 7) NFW to 50 uL
b.Overlap extension PCR
1. Using PrimeSTAR GXL polymerase
Make reaction mix containing:1) 200-300 ng of template 1 (the one that’s bigger in size), 2) Equimolar quantity of template 2, 3) 10 uL GXL buffer (1x) , 4) 4 uL dNTP mix (200 mM each,) 5) 1 uL PrimeSTAR GXL polymerase, 6) NFW to 48 uL, Run for 15 cycles. After that add 1uL each of forward and reverse primers and run for another 15 cycles. Using Pfu DNA polymerase Make reaction mix containing: 1) 200-300 ng of template 1 (the one that’s bigger in size) 2) Equimolar quantity of template 2, 3) 5 uL Pfu buffer (1x) , 4) 4 uL dNTP mix (200 mM each), 5) 2 uL Pfu polymerase, 6) NFW to 48 uL

Run for 15 cycles. After that add 1uL each of forward and reverse primers and run for another 15 cycles.

2 A assembly

RESTRICTION DIGESTION For the Gene insert Make a reaction mixture containing: 1) 100 ng of template, 2) 0.2 uL of EcoR1, 3) 0.2 uL of Pst1, 4) 1 uL of Cutsmart buffer, 5) NFW to 10 uL

For linearized backbone Make a reaction mixture containing: 1) 100 ng of linearized backbone (4 uL), 2) 0.2 uL of EcoR1, 3) 0.2 uL of Pst1, 4) 0.2 uL of Dpn1, 5) 1 uL of Cutsmart buffer, 6) NFW to 10 uL
Incubate both reaction mixtures at 37 deg C for 2 hours and then heat inactivate at 80 deg C for 20 min

LIGATION Make a reaction mixture containing: 1) 2.5 uL of digested backbone(25 ng), 2) 4-6 uL of digested insert (75 ng), 3) 0.5 uL of T4 DNA Ligase, 4) 1 uL of T4 DNA Ligase buffer, 5) NFW to 10 uL
Incubate at 16 deg C overnight and then heat inactivate at 80 deg C for 20 min.

Colony PCR

Mark the number of colonies obtained in the transformed plate (after restriction-ligation). Let’s say we have ‘x’ number of colonies
Make x*10 uL PCR mixtures (10 uL PCR mix for every colony) using Pfu/GXL/Taq polymerase. Aliquot this mixture into 10 PCR tubes.
Pick a part of the colony and crush it inside the respective PCR tube. (Make sure to label your tubes according to your colonies)
Pipette in and out for mixing the solution.
Make another 10 uL PCR reaction mixture for your positive control.
Place all these reaction mixtures in the PCR machine.
After the PCR is complete, load all the samples on a gel of appropriate percentage and run them.
Image the gel to confirm which colonies are positives and are of your interest by comparing with the positive control.
The respective positive colonies are then plasmid extracted and purified for further use.