Team:Groningen/Notebook/19 August 2009
From 2009.igem.org
(→GVP Cluster) |
(→Transporters) |
||
Line 58: | Line 58: | ||
===Transporters=== | ===Transporters=== | ||
+ | |||
+ | HmtA sequencing results came in. 5 out of 7 sequences look nice. 2 of the HmtA vectors were bad with many N. | ||
+ | So far it looks we have the right prefix and start of the gene but it seems it is a Cys mutant. Now we are wondering if we made this mutant or if we got a mutant by accedent (cys-mutants are usualy made for crosslinking experiments in membrane proteins.) | ||
+ | We are asking the people who supplied us with the plasmid and we plan to sequence the source as well. | ||
+ | And time for reverse sequencing to get the other half of the construct. | ||
+ | |||
+ | The PCR's 1 and dirty failed. PCR2 is excised from the gel for downstream processing. | ||
+ | PCR1 is on hold for because of the Cys-mutation. | ||
===Metal Accumulation=== | ===Metal Accumulation=== |
Revision as of 10:49, 19 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
|
---|
Wet
GVP Cluster
- → TODO Restriction for Assembly
- → TODO Gel purification of plasmid/insert
- → TODO Ligation of GVP into vector pSB3K3
- → TODO Transformation of E.coli TOP10 cells (kan.)
- → TODO Test promoter/GVP constructs (grow precultures)
Restriction for Assembly
The vector [http://partsregistry.org/wiki/index.php?title=Part:pSB3K3 pSB3K3] containing the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 low] constitutive promoter was cut with PstI and SpeI to create correct ends for insert of GVP biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 BBa_I750016], which was cut with XbaI and PstI.
- 16μL pSB3K3-L in MQ (0.8μg)
- 2μL Fast digest buffer
- 1μL PstI fast digest enzyme
- 1μL SpeI fast digest enzyme
- 10μL plasmid-GVP in MQ (?μg)
- 6μL MQ (end volume of 20μL)
- 2μL Fast digest buffer
- 1μL PstI fast digest enzyme
- 1μL SpeI/XbaI fast digest enzyme
Restriction was kept at 37C for 30 min. and put on ice until used for gel purification.
Purification
Concentrations
Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
pSB3K3-L (SpeI/PstI) | ? | ? | ? | ? | ? |
GVP (XbaI/PstI) | ? | ? | ? | ? | ? |
Restriction Control
Transporters
HmtA sequencing results came in. 5 out of 7 sequences look nice. 2 of the HmtA vectors were bad with many N. So far it looks we have the right prefix and start of the gene but it seems it is a Cys mutant. Now we are wondering if we made this mutant or if we got a mutant by accedent (cys-mutants are usualy made for crosslinking experiments in membrane proteins.) We are asking the people who supplied us with the plasmid and we plan to sequence the source as well. And time for reverse sequencing to get the other half of the construct.
The PCR's 1 and dirty failed. PCR2 is excised from the gel for downstream processing. PCR1 is on hold for because of the Cys-mutation.
Metal Accumulation
Vectors
Dry
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|
|