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Team:Groningen/project notebook/22 August 2011
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JoyceM1013 (Talk | contribs) (Created page with "{{HeaderGroningen2011}} {{notebookgroningen}} '''Joyce''' <br> <br> After rechecking: constructs with cI-LVA and LasR-LVA are not alright (in total 6 constructs...:( ) <br> Doin...") |
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| + | Week 34: | ||
| + | *hybB promoter measurements | ||
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Joyce
After rechecking: constructs with cI-LVA and LasR-LVA are not alright (in total 6 constructs...:( )
Doing it now over again with definetely the right primer set:
PCR on templates with cI-LVA and LasR-LVA:
Composition per reaction:
10× pfu buffer with MgSO4: 5μl
dNTPs 10mM: 1μl
Forward primer: 1μl
Reverse : 1μl
Template: 1μl
Pfu polymerase: 1μl
MQ water: 40μl
PCR conditions:
Pre heated lid: 111°C
Denaturation: 94°C for 3min
Cycle (33×)
Denaturation: 94°C for 30s
Annealing: 67°C for 30s
Extenstion: 72°C for 2min
Final extension: 72°C for 10min
Store infinite at 4°C
Analyse PCR samples on a 1% agarosegel
Meanwhile:
Purify PCR products and check with nanodrop the DNA concentration
After check, digestion:
------------
PCR did not go well. Do an overnight PCR again and everything from digestion to below will be done tomorrow
PCR on templates with cI-LVA and LasR-LVA:
Composition per reaction:
10× pfu buffer with MgSO4: 5μl
dNTPs 10mM: 1μl
Forward primer: 1μl
Reverse : 1μl
Template: 1μl
Pfu polymerase: 1μl
MQ water: 40μl
PCR conditions:
Pre heated lid: 111°C
Hotstart
Denaturation: 94°C for 5min
Cycle (33×)
Denaturation: 94°C for 30s
Annealing: 69°C for 30s
Extenstion: 72°C for 2min
Final extension: 72°C for 10min
Store infinite at 4°C
Analyse PCR samples on a 1% agarosegel
Digestion
cI-LVA
7μl insert
1μl XbaI
1μl PstI
2μl FD buffer
9μl MQ water
LasR-LVA
6μl insert
1μl XbaI
1μl PstI
2μl FD buffer
10μl MQ water
pSB1A3-DT vector
3μl vector
1μl XbaI
1μl PstI
3μl FD buffer
1μl FastAP
21μl MQ water
Incubate for 60 min at 37 degrees
DNA clean up:
Ligation:
cI-LVA-pSB1A3DT
8.5μl vector
7.1μl insert
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
1.4μl MQ water
LasR-LVA-pSB1A3DT
8.5μl vector
6μl insert
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2.5μl MQ water
Self ligation:
8.5μl vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
8.5μl MQ water
Incubate for 30-40 minutes at room temperature
Transformation
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
Jakub
Week 34:
- hybB promoter measurements
