Oct 21, 2021
in vivo cloning (iVEC)
This protocol is a draft, published without a DOI.
- 1iGEM Gifu
- iGEM Gifu
Protocol Citation: Yuichiroh Ikagawa 2021. in vivo cloning (iVEC). protocols.io https://protocols.io/view/in-vivo-cloning-ivec-bzcmp2u6
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: October 21, 2021
Last Modified: October 21, 2021
Protocol Integer ID: 54381
Abstract
In vivo cloning is a seamless cloning method in which a DNA fragment, to which a homologous sequence has been added by PCR, is introduced directly into Escherichia coli SN1187 for assembly and cloning in the bacterium. The 5' end of the DNA fragment is degraded by Exonuclease III (XthA) from E. coli strain SN1187. The degradation generates cohesive ends, which allow the fragments to hybridize to each other and to be repaired by DNA polymerase. Finally, the nick is repaired by DNA ligase and the plasmid is constructed in the bacterium. Here we describe a protocol for the preparation and transformation of a competent cell, based on the paper and materials by Nozaki et al. [1], [2]
[1]Shingo Nozaki, Hironori Nikia. Exonuclease III (XthA) Enforces In Vivo DNA Cloning of Escherichia coli To Create Cohesive Ends. Journal of Bacteriology, Volume 201, Issue 5, (2019)
[2]iVEC3 株の解説及び使用方法 - SHIGEN
https://shigen.nig.ac.jp/ecoli/strain/download/pdf/strainGeneMutant/iVEC3_jp_20170706.pdf;jsessionid=0282CEF77B5F3B6DF187E89A2867CC05
Materials
Reagents
Escherichia coli SN1187
LB medium
SOC medium
2x TSS
DMSO
Liquid nitrogen
LB agar plate(includes ampicillin/chloramphenicol)
competent cell preparation
competent cell preparation
Scrape the glycerol stock of Escherichia coli strain SN1187 and inoculate it into 3ml of LB medium.
Incubate at 37ºC overnight (16-18 hours) with shaking.
Inoculate 1 ml of the overnight culture into 60 ml of LB medium warmed to 37 ºC.
Incubation at 37 ºC with shaking until reached 0.4-0.5
Chill the medium in ice.
Centrifuge 5,000 g, at 4º C for 5 min.
Discard the supernatant and suspend the pellet in 2 ml of ice-cold LB medium.
Add 1.6 ml of ice-cold 2xTSS solution and mix by pipetting
Add 400 μl of DMSO and mix by pipetting.
Dispense 100 μl each on ice.
Freeze in liquid nitrogen and store at -80 ºC.
Transformation
Transformation
Thaw the 100 μl competent cell E. coli SN1187 strain on ice.
Add 100 ng of DNA samples (vector and insert) into the competent cell.
Mix by gently pipetting and incubate for 20 minutes on ice.
Add 1 ml SOC medium and mix by carefully inverting
Incubate at 37℃ for 1 hour.
Centrifuge at 5,000 g for 1 minute at room temperature
Discard 1 ml of the supernatant.
Resuspend cells with the remaining supernatant by Voltex
Spread the whole culture on an agar plate with the antibiotics (ampicillin/chloramphenicol) for selection.
Incubate overnight at 37°C.