Team:UT-Tokyo/Data/Method

From 2011.igem.org

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==Strains==
==Strains==
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===<span style="text-decoration:underline;">JM109</span>===
+
===<span style="text-decoration:underline;">JM109</span> Tkara Bio INC.===
 +
 
 +
*Genotype:
 +
:recA1, endA1, gyrA96, thi-1, hsdR17(rK-mk+), e14–(mcrA–), supE44, relA1, Δ(lac-proAB)/F' [traD36, proAB+, lac Iq, lacZΔM15]
===BL21===
===BL21===

Revision as of 14:10, 22 September 2011

Reagents

  • SOB broth
reagents final conc. amount
Bacto trypton 2% 20g
NaCl 0.05% 0.5g
Yeast extracs 0.5% 5g
KCl (250mM) 2.5mM 10ml
MilliQ - 1000ml
Total 1L

Before use, add 10ml Mg sol.

Mg sol.
reagents final conc. amount
MgCl2(H2O)6 1M 20.33g
MgSO4(H2O)7 1M 24.648g
MilliQ - 100ml
Total 100ml


  • 20× M9 medium
reagents final conc. amount
Na2HPO4 - 6.0g
KH2PO4 - 3.0g
NaCL - 0.5g
NH4Cl - 1.0g
MilliQ - 50ml
Total 50ml

After A.C. , add following reagents to 1L M9 medium

reagents final conc. amount
1M MgSO4 - 1.0ml
2M Glucose - 5.6ml
1% Thiamine - 1.0ml
1M CaCl2 - 0.1ml
  • LB broth
reagents final conc. amount
Bacto trypton 1% 1g
NaCl 0.5% 0.5g
Yeast extracs 0.5% 0.5g
MilliQ - 100ml
Total 100ml


  • 50× TAE
reagents final conc. amount
Tris 2M 242g
CH3COOH 1M 57.1mL
EDTA (0.5M, pH=8.0) 0.05M 100ml
MilliQ - 1000ml
Total 1L



  • TB
reagents final conc. amount
KOH 500mM sol. 250mM 242g
PIPES 500mM sol. 10mM 2ml
CaCl2 750mM sol. 15mM 2ml
KCl 2.5M sol. 250mM 10ml
MnCl2 550mM sol. 55mM 10ml
MilliQ - 100ml
Total 100ml

Strains

JM109 Tkara Bio INC.

  • Genotype:
recA1, endA1, gyrA96, thi-1, hsdR17(rK-mk+), e14–(mcrA–), supE44, relA1, Δ(lac-proAB)/F' [traD36, proAB+, lac Iq, lacZΔM15]

BL21

ccdB survival (invitrogen)

derived from the TOP10 strain, and offer the following features:

-Resistance to the ccdB gene product
-Resistance to T1 and T5 phage (tonA)
-Cleaner preparations of DNA and better results in downstream applications due to the elimination of non-specific digestion by endonuclease I (endA1)
-Reduced occurrence of non-specific recombination in cloned DNA (recA1)
  • Genotype:
F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG fhuA::IS2

cheZ-

Assembly parts

Digest

Ligation

colony PCR

Transformation

Making Competent E. coli cell

Transformation of E. coli

Purification of DNA

Miniprep

Gel extraction

PCR clean-up

Ethanol precipitation

Analysis of DNA

Gel electrophoresis

Sequencing

Dual luciferase assay

Cell diffsion assay

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