Nov 07, 2022

Public workspaceVector Linearization

This protocol is a draft, published without a DOI.
  • 1Harvard
Felipe FE Edaes
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Protocol Citation: Felipe FE Edaes 2022. Vector Linearization . protocols.io https://protocols.io/view/vector-linearization-civ8ue9w
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
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

Equipment
NanoDrop™ One UV-Vis Spectrophotometer
NAME
spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU
https://www.thermofisher.com/order/catalog/product/ND-ONE-W
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
Measure DNA Concentration of the Plasmid
Use Amount1.0 µL to Amount1.5 µL of the elution buffer (used to elute the DNA) as blank.

Measure blank once to confirm the accuracy.
To measure DNA sample, add Amount1-1.5 µL (equivalent to blank)
Calculate the amount of Plasmid to be used
Calculate the amount of Plasmid to be used
If Amount1 µg is Amount1000 ng , this value should be divided by the amount of DNA measured.
If the required amount is Amount3 µg and the measured sample was Concentration450 ng/μL , the proper calculation should be: 3000/450 = ~6.67 = Amount7.00 µL

Vector Linearization
Vector Linearization
3h
3h
Amount3 µg Plasmid DNA

Amount1 µL Enzyme A

Amount1 µL Enzyme B

Amount2 µL 10X Buffer

Remaining H2O Amount20 µL (up to)

Incubate the reaction for Duration03:00:00 at Temperature37 °C , then place it TemperatureOn ice .

3h
Place it TemperatureOn ice and add Amount0.5 µL phosphatase to the linearized vector.