Nov 07, 2022
Vector Linearization
This protocol is a draft, published without a DOI.
- 1Harvard
Protocol Citation: Felipe Edaes 2022. Vector Linearization . protocols.io https://dx.doi.org/
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: In development
We are still developing and optimizing this protocol
Created: November 06, 2022
Last Modified: November 07, 2022
Protocol Integer ID: 72352
Keywords: vector linearization, protocol for the linearization, linearization, obtained vector
Abstract
Protocol for the linearization of a previously obtained vector.
Guidelines
Add phosphatase to remove phosphate, which remains from the end of linear vectors, thus preventing cells from relinearizing the vectors.
Equipment
Equipment
NanoDrop™ One UV-Vis Spectrophotometer
NAME
spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU
LINK
Sample Volume (Metric): Minimum 1µL; Spectral Bandwidth: ≤1.8 nm (FWHM at Hg 254 nm); System Requirements: Windows™ 8.1 and 10, 64 bit; Voltage: 12 V (DC); Wavelength Range: 190–850 nm
SPECIFICATIONS
Measure DNA Concentration of the Plasmid
Use 1.0 µL to 1.5 µL of the elution buffer (used to elute the DNA) as blank.
Measure blank once to confirm the accuracy.
To measure DNA sample, add 1-1.5 µL (equivalent to blank)
Calculate the amount of Plasmid to be used
If 1 µg is 1000 ng , this value should be divided by the amount of DNA measured.
If the required amount is 3 µg and the measured sample was 450 ng/μL , the proper calculation should be: 3000/450 = ~6.67 = 7.00 µL
Vector Linearization
3h
3 µg Plasmid DNA
1 µL Enzyme A
1 µL Enzyme B
2 µL 10X Buffer
Remaining H2O 20 µL (up to)
Incubate the reaction for 03:00:00 at 37 °C , then place it On ice .
3h
Place it On ice and add 0.5 µL phosphatase to the linearized vector.