Competent Cell Preparation
Inoculate 5ml Luria Bertani(LB) with a colony of the respective strain and let it grow overnight at 37°C at 220rpm.
The next day, inoculate 200ml Super Optimal Broth (SOB) medium with 1ml of the overnight culture and let it grow at 16°C with shaking at 200rpm until it reaches an OD600 of 0.6. Save the media as an OD blank.
Cool the cells on ice for 10mins.
Transfer the culture sequentially to 50ml falcon tubes and spin at 3220 x g for 10mins at 4°C.
Drain the medium and re-suspend each pellet in 80ml ice cold TB .
Place on ice for 10mins.
Spin at 3220 x g for 10mins at 4°C.
Drain the medium and re-suspend each pellet first in 19.8ml of ice cold TB and combine the 2 tubes.
Add 1.4ml of DMSO (Total volume to 40ml ).
Re-suspend each pellet in 20ml of cold TB-DMSO mixture.
Incubate on ice for 10mins.
Transfer 100μl competent cells in each 1.5ml pre-labelled Micro Centrifuge Tubes(MCT) and flash freeze immediately.
Store them at -80°C deep freezer.
Plasmid Isolation by Alkali Lysis Method
Inoculate 2mL of LB medium containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking at 200rpm.
Pellet the cells by centrifugation at 11,000 x g for 1min at 4°C using cooling centrifuge.
Decant the supernatent.
Lysis of Cells
Re-suspend the bacterial pellet in 100µL of ice-cold Alkaline Lysis Solution I by vigorous vortexing.
Add 200µL of freshly prepared Alkaline Lysis Solution II to each bacterial suspension. Mix the contents by inverting the tube rapidly five-ten times. Incubate the suspension on ice for 2-5mins.
Add 150µL of ice-cold Alkaline Lysis Solution III. Close the tube, and disperse Alkaline Lysis Solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube for 3–5mins on ice.
Centrifuge the bacterial lysate at maximum speed for 5min at 4°C in a microcentrifuge. Transfer the supernatant to a fresh tube.
Add an equal volume of phenol:chloroform. Mix the organic and aqueous phases by vortexing, and then centrifuge the emulsion at maximum speed for 2mins at 4°C.
Transfer the aqueous upper layer to a fresh tube.
Recovery of Plasmid DNA
Precipitate the nucleic acids from the supernatant by adding 2 volumes of 100% ethanol at room temperature. Mix the solution by vortexing, and then allow the mixture to stand for 2min at room temperature.
Precipitate nucleic acids by centrifugation at maximum speed for 5mins at 4°C in a microcentrifuge.
Remove the supernatant by gentle aspiration.
Add 1mL of 70% ethanol to the pellet, and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2mins at 4°C in a microcentrifuge.
Again remove all the supernatant by gentle aspiration.
Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (10mins).
Dissolve the nucleic acids in 50µL of TE (pH 8.0) containing 20µg/mL DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few minutes. Store the DNA solution at −20°C.
Bacterial Transformation Protocol
Thaw required number of tubes containing 100μl competent cells on ice.
Add 1-5μl of (1 to 10ng) DNA to the cells. Move the pipette through the cells while dispensing. Gently tap tube to mix.
Incubate cells on ice for 30mins.
Heat-shock cells for 1min in a 42°C water bath; do not shake.
Place on ice for 5mins recovery.
Add 0.9ml SOC Medium (room temperature) to the cells.
Incubate at 37°C with continuous shaking at 225rpm for 1hr.
Centrifuge the culture at 11,000 x g for 2mins.
Remove the supernatent by decanting without disturbing the cell pellet.
Re-suspend cell pellet in residual SOC and spread this culture on LB plates with appropriate antibiotic.
Incubate overnight at 37°C.
Yeast Transformation Protocol by LiAc Treatment
Inoculate yeast cells from plates into 5mL of YPD medium in a culture tube.
Grow overnight with shaking at 200rpm at 30°C.
Pellet cells gently by centrifuging at 6000rpm for 1min.
Wash twice with MilliQ water.
Resuspend in 0.5mL of 1x TE-LiAc solution and rotate at 30°C for 30mins.
Add 5µL of 10mg/ml Salmon Sperm DNA in sterile microcentrifuge tubes designated for transformation and for a negative control.
Add 0.5µg of Plasmid DNA (to be transformed) and 20µL of competent cells into each tube and then vortex.
Add 130µL of freshly prepared PEG-TE-LiAc solution, vortex, and incubate at 30°C for 30mins.
Heat shock for 40mins at 42°C in a water bath.
Spin for 1min at 12000rpm and discard the supernatant.
Re-suspend the cell pellet in 20uL sterile water and spread on appropriate plates.
Incubate plates at 30°C for 2 days.
Restriction Digestion
| Component |
Amount |
| CutSmart Buffer |
1ul |
|
Template
|
1ug |
| Restriction Enzyme |
~1 unit |
|
MilliQ Water
|
Add to make volume 10ul
|
Incubate for 3hrs at appropriate temperature (as recommended by NEB) for digestion confirmation and overnight at appropriate temperature (as recommended by NEB) for cloning.
Ligation
| Component |
Amount |
|
T4 DNA Ligase Buffer
|
1uL |
| Vector |
~10 ng
|
| Insert |
~30 ng (3 times the amount of Vector)
|
|
T4 DNA Ligase
|
1 unit
|
MilliQ water
|
Add to make volume 10ul
|
Incubated at 16°C overnight or at room temperature for 20 mins.
PCR
| Component |
Amount |
| 5x HF Buffer |
10ul |
|
dNTPs (2.5mM)
|
4uL |
|
Forward Primer(10uM)
|
1uL |
|
Reverse Primer(10uM)
|
1uL
|
|
Template DNA
|
~10ng
|
|
Phusion DNA Polymerase
|
1 unit
|
|
MilliQ Water
|
Add to make volume 50ul
|
Specifications:
Step
|
Temperature
|
Time
|
| Initial Denaturation |
98°C |
1 minute |
| 30 Cycles |
98°C
52°C
72°C |
10seconds
15 seconds
10 seconds |
| Final Extension |
72°C |
7 minutes |
* run for 30 cycles
Yeast Plasmid Rescue Protocol:
Inoculate yeast in 5ml YPD medium and incubate at 30°C overnight with shaking at 200rpm.
Spin down 1.5ml culture at 5000rpm for 2 minutes.
Discard supernatant and add 250μl of Favorgen Buffer FADP1 and 200μl of glass beads to each tube.
Beat cells for 5mins.
Add 250μl of FADP2 Buffer and mix by vigorously inverting the tubes.
Add 350μl of N3 and immediately mix by gentle inversion.
Centrifuge for 10mins on full speed.
Transfer supernatant to FavorgenPrep Spin column.
Centrifuge columns for 1min on full speed and discard the flow through.
Wash column with 400μl W1 Wash Buffer followed by 750ul of Buffer PE.
Discard flow through and centrifuge the empty column again for 3 minutes to remove residual wash buffer.
Place column in a fresh recovery tube and add 40μl of Elution Buffer and incubate at RT for 5mins.
Centrifuge the tube at 14,000rpm for 2mins. Discard column. Flow through obtained is the plasmid of interest.
Agarose Gel Preparation
For 1% Agarose gel preparation: Dissolve Agarose in 1x TAE Buffer by boiling and add Ethidium Bromide (5μl/100ml agarose solution).
Pour the gel and let it polymerize until it is solid.
Add appropriate amount of 6x Purple Loading Dye (NEB) to the DNA sample to be loaded.
Place the gel in the tank and add 1x TAE running buffer so that the gel is fully immersed.
Pipette the DNA into wells.
Run the gel at 100 V for 40mins.
Visualize the gel under UV light.
Glycerol Stocks
Prepare a 80% glycerol solution diluted in H2O and autoclave it.
Mix 500μl of overnight culture with 500μl of 80% glycerol solution, and store it at -80◦C.
DNA Preparation by Kits
For DNA preparation FavorPrep™ (Plasmid Extraction Mini Kit) and FavorPrep™ (GEL/PCR Purification Mini Kit) were used.
SOEing PCR Protocol 1
Step 1
- 100ng of the bigger DNA fragment and equimolar concentration of the smaller
fragment was taken in the reaction mixture for PCR.
- The reaction mixture was made as follows:
| Component |
Amount |
|
5x Phusion Buffer
|
2.5µl
|
|
dNTPs (2.5mM)
|
2µl |
|
Larger DNA Fragment
|
~100ng
|
|
Smaller DNA Fragment
|
Equimolar concentration
|
|
Phusion Polymerase HF
|
0.5 unit
|
|
MilliQ Water
|
Add to make volume 25ul
|
Specifications:
Step
|
Temperature
|
Time
|
| Initial Denaturation |
98°C |
1 minute |
| 15 Cycles |
98°C
Annealing Temperature
72°C |
10seconds
15 seconds
30 seconds |
| Final Extension |
72°C |
7 minutes |
* run for 15 cycles
Step 2
1.5µl of the reaction mixture obtained after Step 1 to be used as template for the PCR reaction in Step 2:
| Component |
Amount |
|
5x Phusion Buffer
|
10ul |
|
dNTPs (2.5mM)
|
4uL |
|
Template
|
5uL |
|
Forward Primer
|
2.25uL
|
|
Reverse Primer
|
2.25ul
|
|
Phusion Polymerase HF
|
1 unit
|
|
MilliQ Water
|
Add to make volume 50ul
|
Specifications:
Step
|
Temperature
|
Time
|
| Initial Denaturation |
98°C |
1 minute |
| 35 Cycles |
98°C
Annealing Temperature
72°C |
10seconds
15 seconds
30 seconds |
| Final Extension |
72°C |
7 minutes |
* run for 35 cycles
Soeing PCR Protocol 2
Touchdown PCR
| Component |
Amount |
|
GC Buffer
|
10ul |
|
dNTPs (2.5mM)
|
4uL |
|
Larger Fragment
|
1uL |
|
Smaller Fragment
|
0.3ul
|
|
Forward Primer
|
1ul
|
|
Reverse Primer
|
1ul
|
|
Phusion Polymerase HF
|
0.5 unit
|
|
MilliQ Water
|
Add to make volume 50ul
|
Specifications:
| Temperature |
Time |
|
98°C
|
1 minute
|
|
98°C
|
10 seconds*
|
| 70°C-->50°C |
30 seconds*
|
| 72°C |
30 seconds*
|
|
72°C
|
7 minutes
|
* run for 35 cycles
Sample Preparation for Western blot
Grow cultures overnight to an O.D.600 of around 2.
Centrifuge at 5000 rpm for 5 mins and discard supernatant.
Wash pellet with 1 ml ddH20 and centrifuge at 500 rpm for 5 mins.
Discard supernatant and add 1 ml of 20% TCA and vortex.
Centrifuge at 6000 rpm for 5 mins and discard supernatant.
Add 200ul of 20% TCA and an equal volume of glass beads.
Vortex for 10 secs and then put the tube on a multi vortexer for 15 mins at high speed.
Puncture the tube with a hot needle at the bottom and place on a fresh labelled tube.
Centrifuge at 2000 rpm for 1 min.
Discard the tube with glass beads and vortex the fresh tube for about 10 secs.
Centrifuge at 7000 rpm for 10 mins.
Discard supernatant and add 800 ul of 0.5M tris-cl (pH 7.5) and vortex to mix.
Centrifuge at 7000 rpm for 5 mins.
Discard supernatant as quickly as possible and add 200ul of 0.5M tris-cl (pH 7.5) and 60ul of 6X loading dye with SDS added.
Vortex to mix and boil at 100 degrees Celsius for 10 mins.
Vortex at max speed for 15 mins and run 15 ul of supernatant in an acrylamide gel.
For protocol for preparation of SDS Gel click here.
Sample Preparation for Western blot
Load 15 μL of sample into sample wells using gel-loading pipet tips on SDS gel.
Connect the Power supply to the gel electrode and apply a voltage (90 mV) and then when the dye reaches the separating gel increase the voltage to 120 mV.
Continue electrophoresis until tracking dye moves to the bottom of the gel.
Carefully transfer gel on nitrocellulose filter paper using transfer buffer by running on 90V for 2 hours at 4-degree Celsius.
Place blot in TBST for 5 mins and then add BSA (2.5%) and keep it on a rocker for 1 hr.
Carefully place the membrane in a pack and add primary antibody (1:500,1:1000), while sealing it make sure the all the bubbles are removed.
Then the pack was kept on a rocker for 2 hr and then remove the primary antibody. Wash the blot by keeping it on the rocker with TBST for 7 min. Repeat this step twice.
Now again keep the Blot carefully on the pack and add secondary antibody (1:10,000) while sealing it make sure the all the bubbles are removed. This was kept on a rocker for another 45 min without exposing it to light.
Wash the blot by keeping it on the rocker with TBST for 7 min. Repeat this step twice.
After this Keep the blot for drying for 3 hr and then acquire image according to the antibody used.
