Background
Luria-Bertani (LB) broth is the most widely used medium for the growth of bacteria.
Materials
All weights are per Liter optimize it according to your needs
Add Agarose only on the media that will be used for plates
Label the bottom of plates with antibiotic and date and seal with parafilm
Store the flasks containing LB media and the agar plates at 4°C.
Protocol
Estimated total time: 60-70 Minutes
Background
SOC media (Super Optimal Broth with Catabolic repressor) for competent cell test and bacterial transformation.
Materials
Prepare the following for 1L SOC media
- 250mM KCL by dissolving 1.86g in 100mL dH2O and take from solution 10mL
- 2M MgCl2 by dissolving 19g in 90mL dH2O and take 5mL
- 1M Glucose by dissolving 18g in 90mL dH2O and take 20mL
Protocol
Estimated total time: 120 Minutes
Background
The prerequisite for bacteria to undergo transformation is its ability to take up free, extracellular genetic material. Competent cells are more likely to incorporate foreign DNA if their cell walls are altered so that DNA can pass through more easily. Cells are made competent by a process that uses calcium chloride and heat shock.
Materials
- Once you enter the lab open the Ice maker to chill the falcons and cryovials
- Be sure that the solutions are prepared and put them on ice ALL THE TIME
- Bring the Material altogether ,Clean the bench and start working
Protocol
Estimated total time: 240 Minutes (4 hrs)
Background
Transformations are essential to using the DNA Distribution Kits: resuspend the DNA sample in a well, transform the DNA into competent cells, and select single colonies.
When transforming competent cells, both timing and temperature are very important. Use a lab timer, follow the incubation temperatures closely, and keep materials on ice when required.
Materials
- First thing to do is to pre-chill the eppendorfs , make sure the ice maker is working
- Adjust the water bath to 42 THIS IS CRITICAL
- Pre-warm the SOC and the plates before use
Protocol
Estimated total time: 2 hours (plus 14-18 hour incubation)
We tried this protocol several times and performed changes in the steps like:
- Using different nourishment media such as SOC and LB
- Using different volumes of those nourishment media (200 µl, 950µl)
- Performing different incubation time (45 mins, 1 hr, 2 hrs), and also
heat shock time (40 sec, 45 sec, 1 min, 1.5 mins)
But unfortunately we couldn’t get single transformed cells.
Materials
Reagents
- Add 12 ml Ethanol(96-99%) to washing buffer.
- add 0.8 mgRNase A to neutralization buffer.
Background
Agarose concentrations mainly depend on the length of the loaded DNA.
Materials
Reagents
1x TAE buffer
Protocol
We digested inserts and vectors (pGEX-4T-1) using EcoRI and PstI (Thermo Scientific)
First, gBlocks (inserts) were resuspended in nuclease-free water following these steps
Before opening the tube, spin it down in a microcentrifuge for 3–5 seconds to ensure the DNA is in the bottom of the tube. The pellet can become statically charged and, without this step, can either fly out of the tube or remain in the cap, resulting in loss of yield.
- Add molecular grade water, or a buffer such as IDTE, to reach a final concentration of 10 ng/µL. Our experiments have shown that storage concentrations <1 ng/µL result in loss of material due to adherence to the plastic tube in the absence of a carrier such as tRNA.
- Vortex briefly.
- Incubate at approximately 50°C for 15–20 min.
Heating the tube will ensure the solvent comes in contact with the tiny pellet, even if it is stuck to the side of the tube. Thus, this step will increase the likelihood that the entire pellet will be resuspended.
- Briefly vortex and centrifuge.
gBlocks were resuspended in 20 µL only to reach final concentration 50 ng/µL.
Buffer: O buffer (Orange) is the most suitable buffer for both enzymes.
Buffer working conc. : 1X
Incubation temperature: 37°C
Incubation time: 1-16 hours (we tried 1 hr which resulted in partial digestion, but both 6 hr and overnight were appropriate incubation times for complete digestion)
PstI is not inactivated by incubation at 80°C for 20 min.
There are many options to deactivate PstI:
- Extract with phenol/chloroform.
- Use PCR-Purification kit
- Load directly to the gel electrophoresis.
We ligated inserts and vectors using T4 DNA Ligase (Jena Bioscience)
There are 2 buffers that can be used in this ligation reaction:
Standard Ligation Buffer, 10x conc.
500 mM Tris-HCl pH 7.8 at 25 °C, 100 mM MgCl2, 100 mM DTT, 10 mM ATP and 25 µg/ml BSA
Fast Ligation Buffer, 2x conc.
60 mM Tris-HCl pH 7.8 at 25 °C, 20 mM MgCl2, 20 mM DTT, 2 mM ATP and 10 % PEG
We went through the fast ligation reaction, so we performed the following steps:
Then incubate for 1 hr at 23 °C.
Background
Production of proteins with Cell-Free kit from Arbor Biosciences.
Protocol
Estimated total time:30 Minutes without incubation time
Subtitle
YPD media with artificial seawater for preparing plates
Background
YEPD or yeast extract peptone dextrose, also often abbreviated as YPD.
YPD medium is a complex medium often used for growing S. cerevisiae. It is composed of yeast extract (for vitamins and other nutrients), peptone (broken down proteins), and dextrose (the energy source for the yeast). After the solution is prepared, it is autoclaved so that it is sterile when used.
Materials
Reagents
Protocol
Estimated total time:30 Minutes without incubation time
Materials
Reagents
- Add 12 ml Ethanol(96-99%) to washing buffer.
- add 0.8 mgRNase A to neutralization buffer.
