"
Team:Groningen/project notebook/10 August 2011
From 2011.igem.org
(Difference between revisions)
JoyceM1013 (Talk | contribs) (Created page with "{{HeaderGroningen2011}} {{notebookgroningen}} '''Joyce''' <br> <br> colony PCR of PBAD-pSB1C3: <br> Composition master mix: <br> 10× Taq buffer: 40μl <br> dNTPs 10mM: 8μl <br...") |
|||
| Line 1: | Line 1: | ||
{{HeaderGroningen2011}} {{notebookgroningen}} | {{HeaderGroningen2011}} {{notebookgroningen}} | ||
| + | |||
| + | =Wed 10 August 2011= | ||
| + | '''Vessa''' | ||
| + | ==To Do List== | ||
| + | *Transformation---2nd batch (ON ligation mixture) | ||
| + | *Screening transforman ---Growth ON culture: | ||
| + | **12 variant autoinducing construts | ||
| + | **RFP internal control --> PCR colonies screening | ||
| + | **construct 21.12 (pSB1K3 revDT+P(LasR)+LasR-TAG) | ||
| + | |||
| + | '''Implementation''' | ||
| + | RBS-RFP PCR colonies screening --> pick 9 single clones | ||
| + | <br> 15.5 ul MQ | ||
| + | <br> 2 ul buffer | ||
| + | <br> 1.2 ul MgCl2 | ||
| + | <br> 0.4 ul dNTPs | ||
| + | <br> 0.4 ul Forward | ||
| + | <br> 0.4 ul Reverse | ||
| + | <br> 0.1 ul Taq DNA pol | ||
| + | <br> DNA sample | ||
| + | |||
| + | Growth ON culture for plasmid isolation from | ||
| + | <br> 7 plates 22.xx : 2 clones each | ||
| + | <br> 1 plate 21.12 : 2 clones | ||
| + | |||
| + | '''Results''' | ||
| + | None of growing clones have RBS-RFP insert --> self-ligation/incomplete vector digestion | ||
| + | |||
'''Joyce''' | '''Joyce''' | ||
<br> | <br> | ||
Revision as of 11:53, 21 September 2011
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Wed 10 August 2011
Vessa
To Do List
- Transformation---2nd batch (ON ligation mixture)
- Screening transforman ---Growth ON culture:
- 12 variant autoinducing construts
- RFP internal control --> PCR colonies screening
- construct 21.12 (pSB1K3 revDT+P(LasR)+LasR-TAG)
Implementation
RBS-RFP PCR colonies screening --> pick 9 single clones
15.5 ul MQ
2 ul buffer
1.2 ul MgCl2
0.4 ul dNTPs
0.4 ul Forward
0.4 ul Reverse
0.1 ul Taq DNA pol
DNA sample
Growth ON culture for plasmid isolation from
7 plates 22.xx : 2 clones each
1 plate 21.12 : 2 clones
Results None of growing clones have RBS-RFP insert --> self-ligation/incomplete vector digestion
Joyce
colony PCR of PBAD-pSB1C3:
Composition master mix:
10× Taq buffer: 40μl
dNTPs 10mM: 8μl
MgCl2: 24μl
Forward biobrickvector primer: 8μl
Reverse biobrickvector primer: 8μl
Taq polymerase: 2μl
MQ water: 310μl
PCR conditions:
Pre heated lid: 111°C
Denaturation: 94°C for 10min
Cycle (33×)
Denaturation: 94°C for 30s
Annealing: 60°C for 30s
Extenstion: 72°C for 30s
Final extension: 72°C for 10min
Store infinite at 4°C
Analyse PCR samples on a 1% agarosegel
Plasmid prep of overnight culture PBAD-RBS-GFP colonies 5 and 6
Measure DNA concentration: 40 ng/microliter
Samples were send for sequencing and glycerol stocks were made
dsDNA arrived!!:)
structure in plasmid: SacI-prefix-taRNA-suffix-BamHI-prefix-crRNA-suffix-SalI-prefix-PRM-suffix-HindIII.
Dissolve the dsDNA in 50 microliter of MQ water, pipet 10 times up and down and incubate it for one hour at room temperature
Than make a dilution 100× and use 1 microliter for transformation:
Transformation dsDNA
Add to 40μl competent cells 10μl ligation mixture
Incubate for 30 min on ice
Heat shock: 45s at 42 degrees
Incubate cells on ice for 2 min
Add 1ml LB medium+ 25mM glucose
Incubate cells for 1h/1.5h at 37 degrees
Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
Put the plates in the stove at 37 degrees overnight
Make overnight culture of the PBAD-pSB1C3 colonies 1 and 2
