IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/08/16

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Autocreate 2012/08/16 Entry for IGEM:Paris_Bettencourt_2012/Notebooks/suicide_group/day_by_day/)
(Entry title)
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
-
==Entry title==
+
==Transformation results (from the day before)==
-
* Insert content here...
+
* pSG.008: 1 colony
 +
* pSG.021: 4 colonies
 +
* pSG.017, pSG.018: lots of colonies
 +
* pSG.006: no colony
 +
==Colony PCR==
 +
We did colony PCR for all the colonies of pSG.008 and pSG.021
 +
 +
Colony+50μL H<sub>2</sub>O:
 +
*10μL for cell culture (in 10mL LB+antibiotic)
 +
*boil the rest for 5min, take 1μL for as template DNA
 +
 +
===Protocol===
 +
 +
 +
{| 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;"| H2O
 +
| style="border:0.002cm solid #000000;padding:0.097cm;"| 36.5 uL
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 5x Phusion HF Buffer
 +
| 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;"| 10 uL
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 10 mM dNTPs
 +
| 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;"| Primer mix
 +
| 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;"| Template DNA
 +
| 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;"| Phusion DNA polumerase
 +
| 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;"| 0.5 uL
 +
 +
|}
 +
 +
===PCR Program===
 +
 +
{| 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;"| Cycle step
 +
| 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;"| Temperature (C)
 +
| 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;"| Time
 +
| style="border:0.002cm solid #000000;padding:0.097cm;"| Cycles
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Initial denaturation
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 98
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 30s
 +
| 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:none;padding:0.097cm;"| Denaturation
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 98
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 5-10s
 +
| 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;"| 30*
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Annealing
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 56
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 10-30s
 +
| 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;"| 30*
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Extension
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 72
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 1m
 +
| 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;"| 30*
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Final extension
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 72
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 5-10m
 +
| 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: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;"| 4
 +
| 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
 +
 +
|}
 +
 +
==Ligation of pTet==
 +
We ligated pTet (annealing product) with pSG.002 (E,S)
 +
 +
===Protocol===
 +
 +
{| 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.002 (-) control
 +
| style="border:0.002cm solid #000000;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;"| Vector (pSG.002)
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 3 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;"| 6 uL
 +
 +
|-
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| Insert (pSG.006)
 +
| 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;"| 0.5 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;"| 5.5 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;"| Ligase buffer
 +
| 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;"| T4 ligase
 +
| style="border-top:none;border-bottom:0.002cm solid #000000;border-left:0.002cm solid #000000;border-right:none;padding:0.097cm;"| 0.5 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;"| 0.5 uL
 +
 +
|}
 +
 +
Incubate in 20°C (microscope room) for 1h
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 12:40, 17 August 2012

Project name Main project page
Previous entry      Next entry

Transformation results (from the day before)

  • pSG.008: 1 colony
  • pSG.021: 4 colonies
  • pSG.017, pSG.018: lots of colonies
  • pSG.006: no colony

Colony PCR

We did colony PCR for all the colonies of pSG.008 and pSG.021

Colony+50μL H2O:

  • 10μL for cell culture (in 10mL LB+antibiotic)
  • boil the rest for 5min, take 1μL for as template DNA

Protocol

H2O 36.5 uL
5x Phusion HF Buffer 10 uL
10 mM dNTPs 1 uL
Primer mix 1 uL
Template DNA 1 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 of pTet

We ligated pTet (annealing product) with pSG.002 (E,S)

Protocol

PSG.002 (-) control PSG.006
Vector (pSG.002) 3 uL 6 uL
Insert (pSG.006) - 0.5 uL
H2O 5.5 uL 2 uL
Ligase buffer 1 uL 1 uL
T4 ligase 0.5 uL 0.5 uL

Incubate in 20°C (microscope room) for 1h

Personal tools