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

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{| 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;"|
| 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;"| PSG.001 (-) control
| 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;"| PSG.002 (-) control
| 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;"| PSG.005 (-) control
| 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;"| PSG.006
| 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;"| PSG.008
| 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;"| PSG.017
| style="border:0.002cm solid #000000;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;"| Vector (10-15 ng/uL)
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
| 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;"| 2 uL
|-
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Insert
| 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:none;padding:0.097cm;"| -
| 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:none;padding:0.097cm;"| 0.2 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 0.2 uL
| 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;"| 0.2 uL
|-
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 10x T4 DNA Ligase buffer*
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 2 uL
| 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;"| 2 uL
|-
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| T4 DNA ligase*
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 1 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 1 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 1 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 1 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 1 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 1 uL
| 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 uL
|-
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| H2O
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 15 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 15 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 15 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 14.8 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 13 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 14.8 uL
| 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;"| 14.8 uL
|-
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Total
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 20 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 20 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 20 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 20 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 20 uL
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 20 uL
| 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;"| 20 uL
|}
<nowiki>*I mixed them in 8x before taking 3uL for each reaction</nowiki>
Let them in 22°C for 1h
===Transformation===
Using transformation protocol.
Recovery time +/- 1.5h


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Revision as of 03:41, 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 Antibiotic resistance
PSG.001 (-) control PSG.001 (X,P) - PSB3C5 (low to medium copy plasmid) Cm
PSG.002 (-) control PSG.002 (X,P) - PSB4K5 (low copy plasmid) Kan
PSG.005 (-) control PSG.005 (S,P) - pSB3C5 pLac Cm
PSG.006 PSG.002 (X,P) PSG.004 (X,P)* pSB4k5 pTet Kan
PSG.008 PSG.005 (S,P) PSG.014 (X,P) pSB3C5 pLac ColE2 Imm Cm
PSG.017 PSG.001 (X,P) PSG.015 (X,P)* pSB3C5 pConstitutive (0.86) Cm
PSG.018 PSG.002 (X,P) PSG.016 (X,P)* pSB4K5 pConstitutive (0.51) Kan

*Oligos annealing product

Protocol

PSG.001 (-) control PSG.002 (-) control PSG.005 (-) control PSG.006 PSG.008 PSG.017 PSG.018
Vector (10-15 ng/uL) 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL
Insert - - - 0.2 uL 2 uL 0.2 uL 0.2 uL
10x T4 DNA Ligase buffer* 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL
T4 DNA ligase* 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL
H2O 15 uL 15 uL 15 uL 14.8 uL 13 uL 14.8 uL 14.8 uL
Total 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL

*I mixed them in 8x before taking 3uL for each reaction

Let them in 22°C for 1h

Transformation

Using transformation protocol.

Recovery time +/- 1.5h

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