Nov 28, 2022
Plasmid Expansion
- Ali Albalakhi1,2,
- Ning Xia1,2
- 1Massachusetts General Hospital;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Protocol Citation: Ali Albalakhi, Ning Xia 2022. Plasmid Expansion. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jrjjl1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working.
Created: November 28, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 73277
Keywords: ASAPCRN
Abstract
This is the protocol for plasmid expansion.
Attachments
Thaw the competent cells on ice
Chill approximately 5 μg (2 μl) of Plasmid DNA in 1.5 mL microcentrifuge tube
Add 50 μl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA. Do not vortex.
Place the mixture on ice for 30 minutes. Do not mix.
Heat shock at 42°C for 30 seconds*. Do not mix.
Add 950 μl of room temperature media* to the tube.
Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
Warm selection plates to 37°C.
Spread 50–100 μl of the cells and ligation mixture onto the plates.
Incubate overnight at 37°C.
Please note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period,
please follow the recommendations provided by the competent cells’ manufacturer.
Plasmids and antibiotics
Plasmids and antibiotics
| A | B | |
| PLVX mCherry (HLA-A2) | Amp 50μg/mL | |
| Ploc MLANA (MART1) | Blasticidin (50μg/mL) | |
| psPAX2 | Amp 50μg/mL | |
| VSV.G | Amp 50μg/mL |
Preparation of Liquid Media - LB-Ampicillin Broth and LB-Blasticidin Broth:
Preparation of Liquid Media - LB-Ampicillin Broth and LB-Blasticidin Broth:
Add 20g of LB broth Lennox into 1 liter of distilled water in an Erlenmeyer flask
Autoclave the LB broth in the Flask on a wet cycle for 30min
Storage of LB in 4ºC is recommended and is good for up to 1 year
Ensure that the LB broth has come to room temperature before you add ampicillin
The final concentration of ampicillin in the broth should be 50μg/mL
LB broth containing ampicillin should be stored at 4ºC for up to 1 month
Preparation of Solid Media
Preparation of Solid Media
LB-Amp Agar and LB-Blasticidin Agar: Each Petri Dish takes about 10mL of LB-Agar so scale the volume accordingly.
1 LB agar tablet makes 50mL
Take 1 tablet and dissolve it in 50mL of distilled water in an autoclaved flask
Autoclave the LB agar for 30min on a wet cycle
Once the cycle is complete check to make sure that all the agar is dissolved
Allow the LB agar to cool until it is comfortable to the touch (50ºC), a water batch set to 50ºC is useful for this step
While the agar is cooling label the plates using sharpie for each antibiotic
a. Ampicillin = Red
b. Blasticidin = Blue
The final concentration of ampicillin and Blasticidin should be 50μg/mL
Make sure that the LB agar has cooled to 50ºC as excessive heat will degrade the antibiotics