"
Team:Groningen/project notebook/30 August 2011
From 2011.igem.org
(Difference between revisions)
(Created page with "{{HeaderGroningen2011}} {{notebookgroningen}} '''Olesja''' <br> Flowcytometry measurement <br> PHybB induction in RW and Top10 cells <br> cells grown at 27 degree Celsius <br> c...") |
JoyceM1013 (Talk | contribs) |
||
| Line 6: | Line 6: | ||
construct: | construct: | ||
*PHybB_GFP_DT | *PHybB_GFP_DT | ||
| + | <br> | ||
| + | '''Joyce''' | ||
| + | <br> Colony PCR of PBAD-RBS-GFP-DT | ||
| + | <br> | ||
| + | <br> Taq 10× buffer: 20μl | ||
| + | <br> dNTPs 10mM: 4μl | ||
| + | <br> MgCl2: 12μl | ||
| + | <br> Forward Biobrick primer: 4μl | ||
| + | <br> Reverse Biobrick primer: 4μl | ||
| + | <br> Taq DNA polymerase: 1μl | ||
| + | <br> MQ water: 155μl | ||
| + | <br> | ||
| + | <br> PCR conditions: | ||
| + | <br> Preheated lid: 111°C | ||
| + | <br> Denaturation: 94°C for 10 min. | ||
| + | <br> Cycle 33×: | ||
| + | <br> denaturation: 94°C for 30s. | ||
| + | <br> annealing: 60°C for 30s. | ||
| + | <br> Extension: 72°C for 2,5 min. | ||
| + | <br> Final extension: 72°C for 10 min. | ||
| + | <br> Store infinite at 4°C | ||
| + | <br> | ||
| + | <br> Analyse on a 1% TBE agarose gel. | ||
| + | <br> Transformants seem to contain PBADaraC, but not RBS-GFP-DT since the fragment has a size of 1500 a 1600 bp. | ||
| + | <br> | ||
| + | <br> Overnight cultures PhybB-cI-LVA-DT colonies 2 and 3 and PhybB-LasR-LVA- colonies 2 and 6(with RBSes xD)grew. | ||
| + | <br> Plasmid prep was done. | ||
| + | <br> ND1000- Nanodrop measurements results: DNA concentrations were 30-40 ng/μl | ||
| + | <br> Samples were send for sequencing and glycerol stocks were made | ||
| + | <br> Measurements with FACS were done for PhybB-RBS-GFP, Olesja continues. Also PhybB-RBS-GFP-DT in E.coli BW strain | ||
| + | <br> | ||
| + | <br> Protocol obtained again for gDNA isolation with magnetic beads for report | ||
| + | <br> | ||
| + | <br> New cloning strategy for PBAD-cI-LVA-DT and PBAD-LasR-LVA-DT | ||
| + | <br> | ||
| + | |||
{{FooterGroningen2011}} | {{FooterGroningen2011}} | ||
Revision as of 15:15, 20 September 2011
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Olesja
Flowcytometry measurement
PHybB induction in RW and Top10 cells
cells grown at 27 degree Celsius
construct:
- PHybB_GFP_DT
Joyce
Colony PCR of PBAD-RBS-GFP-DT
Taq 10× buffer: 20μl
dNTPs 10mM: 4μl
MgCl2: 12μl
Forward Biobrick primer: 4μl
Reverse Biobrick primer: 4μl
Taq DNA polymerase: 1μl
MQ water: 155μl
PCR conditions:
Preheated lid: 111°C
Denaturation: 94°C for 10 min.
Cycle 33×:
denaturation: 94°C for 30s.
annealing: 60°C for 30s.
Extension: 72°C for 2,5 min.
Final extension: 72°C for 10 min.
Store infinite at 4°C
Analyse on a 1% TBE agarose gel.
Transformants seem to contain PBADaraC, but not RBS-GFP-DT since the fragment has a size of 1500 a 1600 bp.
Overnight cultures PhybB-cI-LVA-DT colonies 2 and 3 and PhybB-LasR-LVA- colonies 2 and 6(with RBSes xD)grew.
Plasmid prep was done.
ND1000- Nanodrop measurements results: DNA concentrations were 30-40 ng/μl
Samples were send for sequencing and glycerol stocks were made
Measurements with FACS were done for PhybB-RBS-GFP, Olesja continues. Also PhybB-RBS-GFP-DT in E.coli BW strain
Protocol obtained again for gDNA isolation with magnetic beads for report
New cloning strategy for PBAD-cI-LVA-DT and PBAD-LasR-LVA-DT
