IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/08/31: Difference between revisions

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| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| hold
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| hold
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| 1
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| 1
|}
==Ligation==
===Products===
{| style="border-spacing:0;"
| style="border-top:0.002cm solid #000000;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Product
| style="border-top:0.002cm solid #000000;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Vector (digested with)
| style="border-top:0.002cm solid #000000;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Insert (digested with)
| style="border:0.002cm solid #000000;padding:0.097cm;"| Description
|-
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.001 (-) control
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.001 (X,P)
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| -
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| PSB3C5 (low to medium copy plasmid)
|-
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.002 (-) control
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.002 (X,P)
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| -
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| PSB4K5 (low copy plasmid)
|-
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.005 (-) control
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.005 (S,P)
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| -
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| pSB3C5 pLac
|-
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.006
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.002 (X,P)
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.004 (X,P)*
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| pSB4k5 pTet
|-
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.008
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.005 (S,P)
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.014 (X,P)
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| pSB3C5 pLac ColE2 Imm
|-
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.017
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.001 (X,P)
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.015 (X,P)*
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| pSB3C5 pConstitutive (0.86)
|-
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.018
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.002 (X,P)
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| PSG.016 (X,P)*
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:0.002cm solid #000000;padding:0.097cm;"| pSB4K5 pConstitutive (0.51)


|}
|}

Revision as of 03:21, 1 August 2012

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PCR of plasmids from Osnat

PCR products

Plasmid Product Product description Primers Length
ColE2 pSG.013 Colicin E2 Toxin iSG.003 + iSG.004 1797 bp
ColE2 pSG.014 Colicin E2 Immunity iSG.005 + iSG.006 314 bp
ColE7 pSG.019 Colicin E7 Toxin iSG.001 + iSG.002 1784 bp
ColE7 pSG.020 Colicin E7 Immunity iSG.007 + iSG.008 317 bp

Primer mix

100 uM forward primer 10 uL
100 uM reverse primer 10 uL
H2O 80 uL

Protocol

Prepared in ice:

H2O 37 uL
5x Phusion HF Buffer 10 uL
10 mM dNTPs 1 uL
Primer mix 1 uL
Template DNA 0.5 uL
Phusion DNA polumerase 0.5 uL

PCR program

Cycle step Temperature (C) Time Cycles
Initial denaturation 98 30s 1
Denaturation 98 5-10s 30*
Annealing 56 10-30s 30*
Extension 72 1m 30*
Final extension 72 5-10m 1
4 hold 1

Ligation

Products

Product Vector (digested with) Insert (digested with) Description
PSG.001 (-) control PSG.001 (X,P) - PSB3C5 (low to medium copy plasmid)
PSG.002 (-) control PSG.002 (X,P) - PSB4K5 (low copy plasmid)
PSG.005 (-) control PSG.005 (S,P) - pSB3C5 pLac
PSG.006 PSG.002 (X,P) PSG.004 (X,P)* pSB4k5 pTet
PSG.008 PSG.005 (S,P) PSG.014 (X,P) pSB3C5 pLac ColE2 Imm
PSG.017 PSG.001 (X,P) PSG.015 (X,P)* pSB3C5 pConstitutive (0.86)
PSG.018 PSG.002 (X,P) PSG.016 (X,P)* pSB4K5 pConstitutive (0.51)