Thu 18 Aug 2011

Vessa

To Do List

1. Ligate pSB1K3+revDT+P(LasR)+LasR and revP(BAD)-DT

• Digest vector with SP, insert with XP--incubate 1 hr 37 degree
• Purify and elute in 50 μl
• Ligate ON

2. Ligate pSB1A3+RBS-RFP-DT and digested Pc

• Digest vector with EX, insert with ES--incubate 1 hr 37 degree
• Run digested insert on gel then extract it from 3% agarose gel
• Purify and elute in 50 ul for vector and 20 μl for insert
• Ligate ON

Implementation

1. Ligate 12 variants of pSB1K3+revDT+P(LasR)+LasR and revP(BAD)-DT
Double digestion
Vector --> SP
18 μl MQ
3 μl 10x buffer
1 μl RE1
1 μl RE2
1 μl FastAP
6 μl vector

insert --> XP
14 μl MQ
2 μl 10x buffer
1 μl RE1
1 μl RE2
1 μl FastAP
2 μl vector

Ligation mix [insert] = 11 ng/nl
For 1:9 vector to insert ratio, insert mass = 100 ng ~ 10 μl
2 μl 10x buffer
1 μl T4 DNA ligase
7 μl vector
10 μl insert

2. Ligate pSB1A3+RBS-RFP-DT and digested Pc
Double digestion
Vector --> EX [vector] = 43 ng/ul
20 μl MQ
3 μl 10x buffer
1 μl RE1
1 μl RE2
1 μl FastAP
4 μl vector

insert --> ES [insert] = 95 ng/ul
11 μl MQ
2 μl 10x buffer
1 μl RE1
1 μl RE2
5 μl vector

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

Highlight

• Weekly team meeting at 2 pm
• Update meeting with supervisor and advisor at 4 pm

Joyce

colony PCR of PBAD-RBS-GFP-DT old and new, PhybB-taRNA-DT 1 and 4 and PhybB-RBS-GFP-DT in TOP10 cells:
Composition master mix:
10× Taq buffer: 84μl
dNTPs 10mM: 16.8μl
MgCl2: 50.4μl
Forward biobrickvector primer: 16.8μl
Reverse biobrickvector primer: 16.8μl
Taq polymerase: 4.2μl
MQ water: 651μl

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 2.5min
Final extension: 72°C for 10min
Store infinite at 4°C
Analyse PCR samples on a 1% agarosegel

PBAD-RBS-GFP-DT (with the new isolated vector) can possibly contain two right clones, colony 7 and 8 since the size is around
2000bp. Colonies 7 and 8 were grown on LB overnight.
Some of the PhybB-taRNA-DT 1 and 4 samples have the right fragment size. Colonies 1.1 and 4.1 were grown on LB overnight.
PhybB-RBS-GFP-DT in TOP 10 cells: all samples have the right fragment size. Colonies 2 and 3 were grown on LB overnight.


Dissolve new ordered primer Forward primer PBAD-araC 623bp
Prepare some samples for sequencing with this primer:
PBADaraC-cI-LVA-DT and PBADaraC-LasR-LVA-DT
Filling in forms for stage
Meeting at 14:00 and update meeting at 16:00