Tue 9 August 2011

Vessa

To do List

• Ligation and transformation --> 12 variants autoinducing loop + RFP internal control

Ligation mix---1 h incubation at RT
2 μl 10X FD buffer
1 μl T4 DNA ligase
vector (20ng)
insert

Implementation

1.revP(BAD)+DT into pSB1K3 revDT+P(LasR)+LasR
[insert] = 700 ng/50 μl = 14 ng/μl
For 1:6 vector to insert ration, insert mass = 62 ng ~ 4.4 μl

12 ligation reaction, each containing:
4.1 μl MQ
2 μl 10x buffer
1 μl T4 DNA ligase
8.5 μl vector
4.4 μl insert

2.RBS-RFP into pSB1A3-DT
[insert] = 222 ng/50 μl = 14 ng/μl
For 1:6 vector to insert ration, insert mass = 40 ng ~ 9 μl

Ligation mix
2 μl 10x buffer
1 μl T4 DNA ligase
8.5 μl vector
9 μl insert

Transformation Protocol

• Add 10 μl ligation mixture to 40μl E.coli DH5alpha competent cells
• Incubate on ice for 30 min
• Heat shock at 42 degrees for 90 sec
• Place tubes back on ice immediately for 10 minutes.
• Add 1 ml LB without antibiotic
• Incubate the culture at 37 degrees for 1 hour
• Plate out the cell on agar plates with the appropiate antibiotic
• Incubate the plates ON at 37 degrees.

Joyce

Colony PCR of yesterday's transformation. Analyse it on a 1% agarosegel
Overnightculture of RBS-GFP-DT grew. Plasmid prep of RBS-GFP-DT overnight culture
Measuring DNA concentration with the ND1000 nanodrop: 32.6ng/μl

Digestion of pSB1C3+ PBAD/araC, RBS-GFP-DT vector and PBAD/araC
RBS-GFP-DT vector:
5μl vector
1μl EcoRI
1μl XbaI
1μl FastAP
3μl FD buffer
19μl MQ water

PBAD/araC:
5μlPBAD/araC
1μl EcoRI
1μl SpeI
2μl FD buffer
11μl MQ water

pSB1C3+PBAD/araC
2μl vector
2μl PBAD/araC
1μl EcoRI
1μl PstI
2μl FD buffer
12μl 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