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Team:Groningen/project notebook/8 August 2011
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*Holidays | *Holidays | ||
| + | '''Christoph''' | ||
| + | Repeated some of the clonings. | ||
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Latest revision as of 00:25, 22 September 2011
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Mon 8 August 2011
Vessa
Main Goals:
- Constrution of autoinducing loops
- Construction of internal control
Wet labwork
Cloning revP(BAD)+DT (insert) into pSB1K3-revDT+P(LasR)+LasR+TAG (vector)
- PCR part --> revP(BAD)+DT from pSB1A3-revP(BAD)-DT using biobrick primer set
- clone into pSB1K3-revDT+P(LasR)+LasR+TAG (12 different variants) on LB+kanamycine plate
- Double digestion --> vector with SP, insert with XP
- Ligate at 4 degrees ON
Cloning RBS-RFP (insert) into pSB1A3-DT (vector)
- PCR part [http://partsregistry.org/Part:BBa_E1010 RBS-RFP] from distributed DNA plasmid using 29F RBS-RFP and BB suffix primer set
- clone to pSB1A3-DT on LB+ampicilin plate
- Double digestion --> vector with EX, insert with ES
- Ligate at 4 degrees ON
PCR Master Mix
37.71 μl MQ
5 μl 10X Taq buffer
3 μl MgCl2 (25mM)
1 μl dNTPs (10mM)
1 μl Forward (10μM)
1 μl Reverse (10μM)
0.25 μl Taq (5U/μl)
0.04 μl Pfu
1 μl Template DNA
50 μl Total reaction
PCR Results
- revP(BAD)+DT --> proper size band!
- RBS-RFP --> proper size band!
Double digestion mix ---incubate ON at 37 degrees
3 μl 10X FD buffer
1 μl enzyme #1
1 μl enzyme #2
1 μl alkaline phosphatase
vector (1μg)/insert (200ng)
add up to 30 μl with MQ
Notes
- ON culture P(hybB)+RBS-GFP+DT --> NOT grow
Dry labwork
- Kanamycine resistance cassete sequences analysis
Joyce
Digestion of pSB1C3+ PBAD/araC, RBS-GFP-DT vector and PBAD/araC
RBS-GFP-DT vector:
3μl vector
1μl EcoRI
1μl XbaI
1μl FastAP
3μl FD buffer
14μl MQ water
PBAD/araC:
5μlPBAD/araC
1μl EcoRI
1μl SpeI
2μl FD buffer
11μl MQ water
pSB1C3+PBAD/araC
1μl vector
1μl PBAD/araC
1μl EcoRI
1μl PstI
2μl FD buffer
14μl MQ water
Digest for 1h at 37 degrees
DNA clean up with PCR clean up kit
Ligation:
PBAD/araC-RBS-GFP-DT
8.5μl vector
5.6μl PBAD/araC
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
2.9μl MQ water
Self ligation RBS-GFP-DT
8.5μl vector
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
8.5μl MQ water
pSB1C3+PBAD/araC
In the DNA clean up step, the DNA was eluted in 17μl for direct use in ligation
so:
17μl pSB1C3+PBAD/araC * EcoRI*PstI purified
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
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
Primer design for PBAD/araC around 600bp of araC for samples PBAD-cI-LVA-DT and PBAD-LasR-LVA-DT
Ordered them via proligo
Grow RBS-GFP-DT vector overnight from glycerolstock
Jakub
Week 32:
- Holidays
Christoph Repeated some of the clonings.
