Team:Groningen/Notebook/17 July 2009
From 2009.igem.org
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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. | 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. | ||
+ | |||
+ | {| | ||
+ | | | ||
+ | <!--Tabel 1 hier--> | ||
+ | |||
+ | {| border="1" | ||
+ | |+ '''MasterMix1''' | ||
+ | ! Component !! Mg/K | ||
+ | |- | ||
+ | ! 2x Phusion MM | ||
+ | | 12.5 uL | ||
+ | |- | ||
+ | ! MQ | ||
+ | | 9.5 uL | ||
+ | |- | ||
+ | ! F mut1 | ||
+ | | 1 uL | ||
+ | |- | ||
+ | ! R mut2 | ||
+ | | 1 uL | ||
+ | |- | ||
+ | ! DNA | ||
+ | | 1 uL | ||
+ | |} | ||
+ | |||
+ | |width="10"| | ||
+ | | | ||
+ | <!--Tabel 2 hier--> | ||
+ | {| | ||
+ | ! PCR1 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 | ||
+ | |} | ||
+ | |width="10"| | ||
+ | | | ||
+ | <!--Tabel 3 hier--> | ||
+ | |} | ||
===Metal Accumulation=== | ===Metal Accumulation=== |
Revision as of 14:17, 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.
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Metal Accumulation
Vectors
Dry
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