Team:Groningen/Notebook/17 July 2009
From 2009.igem.org
(→Transporters) |
(→Transporters) |
||
Line 85: | Line 85: | ||
|- | |- | ||
|Denaturing | |Denaturing | ||
- | | | + | |98° |
- | | | + | |1.30 min |
|- | |- | ||
| | | | ||
- | |Start Cycles | + | |Start Cycles 30X |
|- | |- | ||
|Denaturing | |Denaturing | ||
- | | | + | |98° |
- | | | + | |10 sec |
|- | |- | ||
|Annealing | |Annealing | ||
Line 101: | Line 101: | ||
|Elongation | |Elongation | ||
|72° | |72° | ||
- | |2 | + | |2:10 min |
|- | |- | ||
| | | | ||
Line 118: | Line 118: | ||
<!--Tabel 3 hier--> | <!--Tabel 3 hier--> | ||
|} | |} | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
Line 125: | Line 132: | ||
{| border="1" | {| border="1" | ||
- | |+ ''' | + | |+ '''MasterMix2''' |
! Component !! Mg/K | ! Component !! Mg/K | ||
|- | |- | ||
Line 158: | Line 165: | ||
<!--Tabel 2 hier--> | <!--Tabel 2 hier--> | ||
{| | {| | ||
- | ! | + | ! PCR1 program |
! Temperature | ! Temperature | ||
! Time | ! Time | ||
|- | |- | ||
|Denaturing | |Denaturing | ||
- | | | + | |98° |
- | | | + | |1.30 min |
|- | |- | ||
| | | | ||
- | |Start Cycles | + | |Start Cycles 30X |
|- | |- | ||
|Denaturing | |Denaturing | ||
- | | | + | |98° |
- | | | + | |10 sec |
|- | |- | ||
|Annealing | |Annealing | ||
Line 179: | Line 186: | ||
|Elongation | |Elongation | ||
|72° | |72° | ||
- | |2 | + | |2:10 min |
|- | |- | ||
| | | | ||
Line 196: | Line 203: | ||
<!--Tabel 3 hier--> | <!--Tabel 3 hier--> | ||
|} | |} | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
{| | {| |
Revision as of 14:19, 17 July 2009
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.
- For plasmid isolation the Sigma Plasmid isolation kit was used (eluted in 50ul MQ).
- DNA concentrations:
Sample | ng/ul | 260/280 | 260/230 |
gpv-promoter test (colony 9) | 82.5 | 2.02 | 2.21 |
gpv-promoter test (colony 10) | 71.9 | 1.96 | 2.21 |
- Restriction analysis with SpeI and PstI to find insert length.
- Expected length with RFP insert ~1kb
- Expected length with gvp insert ~7kb
Restriction mixture for J23100+gvp (fragment sizes: 2096 (promoter + plasmid backbone), 887)
- 10 μL promoter plasmid (61.4 ng/μL, 96.1 ng/μL)
- 6μL MQ
- 2μL Fast digest buffer
- 1μL PstI fast digest enzyme
- 1μL SpeI fast digest enzyme
Transporters
Because of the negative results we will try to get positive results with an other polymerase enzyme; Phusion. And try the cloning again as planned. PCR1 Fw,mut1RC should give product of 1153bp and PCR2 Rev,mut2RC a 261bp size product.
|
|
|
|
|
|
Metal Accumulation
Vectors
Dry
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|
|