TITLE: in vivo cloning (iVEC)
AUTHORS: Yuichiroh  Ikagawa

[DESCRIPTION]
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

[STEPS]
SECTION: Transformation
13. Add 100 ng of DNA samples (vector and insert) into the competent cell.
SECTION: Transformation
14. Mix by gently pipetting and incubate for 20 minutes on ice.

SECTION: Transformation
15. Add 1 ml SOC medium and mix by carefully inverting
SECTION: Transformation
16. Incubate at 37℃ for 1 hour.
SECTION: Transformation
17. Centrifuge at 5,000 g for 1 minute at room temperature
SECTION: Transformation
18. Discard 1 ml of the supernatant.
SECTION: Transformation
19. Resuspend cells with the remaining supernatant by Voltex
SECTION: Transformation
20. Spread the whole culture on an agar plate with the antibiotics (ampicillin/chloramphenicol) for selection.
SECTION: Transformation
21. Incubate overnight at 37°C.
21. 
SECTION: Transformation
12. Thaw the 100 μl competent cell E. coli SN1187 strain on ice.
SECTION: competent cell preparation
34. Scrape off the glycerol stock of  Escherichia coli SN1187 and inoculate 3ml of L
LB medium.
SECTION: competent cell preparation
34. Incubate at 37ºC overnight (about 16~18hours) with shaking.
SECTION: competent cell preparation
34. Inoculate 1 ml of the overnight culture into 60 ml of LB medium warmed to 37 ºC.
SECTION: competent cell preparation
34. Incubate at 37 ºC for 90 minutes with shaking.
SECTION: competent cell preparation
34. Transfer the medium onto ice and chill.
SECTION: competent cell preparation
34. Centrifuge 5,000 g at 4 ℃ for 5 minutes and discard the supernatant.
SECTION: competent cell preparation
34. Suspend the pellets in 2 ml of ice-cold LB medium.
SECTION: competent cell preparation
34. Add 1.6 ml of ice-cold 2xTSS solution and mix thoroughly by pipetting.
SECTION: competent cell preparation
34. Add 0.4 ml of DMSO (at room temperature) and mix by pipetting.

SECTION: competent cell preparation
10. Dispense 100 μl each on ice.
34. Freeze in liquid nitrogen and store at -80 ºC
SECTION: competent cell preparation
1. Scrape the glycerol stock of Escherichia coli strain SN1187 and inoculate it into 3ml of LB medium.
SECTION: competent cell preparation
2. Incubate at 37ºC overnight (16-18 hours) with shaking.
SECTION: competent cell preparation
3. Inoculate 1 ml of the overnight culture into 60 ml of LB medium warmed to 37 ºC.
SECTION: competent cell preparation
6. Centrifuge 5,000 g, at 4º C for 5 min.
SECTION: competent cell preparation
7. Discard the supernatant and suspend the pellet in 2 ml of ice-cold LB medium.
SECTION: competent cell preparation
8. Add 1.6 ml of ice-cold 2xTSS solution and mix by pipetting
SECTION: competent cell preparation
9. Add 400 μl of DMSO and mix by pipetting.
SECTION: competent cell preparation
11. Freeze in liquid nitrogen and store at -80 ºC.
SECTION: competent cell preparation
4. Incubation at 37 ºC with shaking until reached 0.4-0.5
SECTION: competent cell preparation
5. Chill the medium in ice.