From 2009.igem.org
Wet
GVP Cluster
Discussion:
All colonies (transformants vector + promoter ligated with gvp) and o/n culture became red, so probably RFP is still in there...
What might be the problem? The vector with promoter self-ligated due to uncomplete digestion (done 15 July 2009) leading to the following fragments:
- vector+RFP linear
- vector
- RFP
When the upper fragment (~2kb) is contaminated with linear vector this would lead high efficiency self-ligation instead of ligating gvp with the emtpy vector.
- →TODO Therefore the restriction/purification/ligation will be redone and selection of non-red colonies will be made!
- →DONE Also plasmid from the o/n cultures will be purified and analysed by restriction analysis.
- Used Sigma Plasmid isolation kit.
- DNA concentrations:
Transporters
MasterMix3
Component | Mg/K
|
MQ
| 28.5 uL
|
F mut1
| 2 uL
|
R mut2
| 2 uL
|
dNTP
| 2 uL
|
MgCl
| 3 uL
|
taq buffer KCl
| 5 uL
|
taq buffer (NH)SO
| 5 uL
|
taq polymerase
| 2.5 uL
|
|
|
PCR 3 program
| Temperature
| Time
|
Denaturing
| 95°
| 2.00 min
|
| Start Cycles 25X
|
Denaturing
| 95°
| 30 sec
|
Annealing
| 55°
| 20 sec
|
Elongation
| 72°
| 2.10 min
|
| End cycles
|
Final elongation
| 72°
| 10 min
|
Hold
| 4°
| Forever
|
|
|
|
Metal Accumulation
Vectors
Dry