Oct 16, 2021
Transformation of E. coli with plasmid
- 13rd year BS-MS, IISER Pune
- iGEM IISER Pune India 2021
Protocol Citation: sanjana 2021. Transformation of E. coli with plasmid. protocols.io https://dx.doi.org/10.17504/protocols.io.byz3px8n
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: October 13, 2021
Last Modified: October 16, 2021
Protocol Integer ID: 54043
Abstract
This protocol can be used to transform electrocompetent E. coli cells with a plasmid.
Preparation
Preparation
30m
30m
Wash cuvette with 100 % (v/v) ethanol and UV in the hood for 00:20:00
20m
Pre-thaw electrocompetent cells and plasmid on wet ice for 00:10:00
10m
Electroporation
Electroporation
20m 40s
20m 40s
Add 1 µL of plasmid DNA to 50 µL electrocompetent cells
Transfer cell-DNA mixture into the cuvette and add equal amount (50 µL ) of MilliQ
Let the cuvette sit in wet ice for 00:20:00
20m
Place cuvette in BioRad pulser and pulse at 2.5 keV
Within 00:00:40 add 100 µL of 2X LB to the cuvette
40s
Incubation
Incubation
30m
30m
Place cuvette in an incubator at 37 °C for 00:30:00
30m
Then use a bent pipette tip to spread the cuvette's contents onto a plate made with LB agar and the appropriate antibiotics. Spread until plate is dry
Place the plate in an incubator at 37 °C Overnight
Make glycerol stocks of transformed cells and store for future use