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Jax (Jackson laboratories) is the biggest (and best) lab animal breeding organization in the world. If you read the notes carefully, you will note that it is a spontaneous mutation and that it was bred by crossing siblings. C57 black mice are very common in many labs around the world.
A permissive background is one that allows expression of the mutation without adverse affects on the animal to a large extent. It may also mean that there is a limited capability to repair the genome, or something similar. I don't know the genetic basis for it at all.
For refs, try the ones starting about 2/3 of the way down the page.
#138510 What does this description of the C57BL/6J mouse strain mean?
Posted
on 29 July 2012 - 12:32 PM
#138907 Alkaline phosphate treatment of vector SalI to ligate it to insert SalI site
Posted
on 03 August 2012 - 11:01 AM
If you need to dephosphorylate your vector, you should do it with the minimal amount of CIP and for the shortest time possible. Switching to shrimp alkaline phosphatase or antarctic phosphatase would be a good idea, so that you can heat inactivate it. Another option is to use PCR to amplify your vector backbone, then treat with DpnI to get rid of parent circular vector. The PCR product will not transform (ideally, you would use different restriction enzymes at both ends, to eliminate the recircularization after cutting and ligating).
You didn't say how you know your cells are competent (and, equally important, how competent).
You didn't say how you know your cells are competent (and, equally important, how competent).
#138969 pkD46 vector growth in E.coli problem
Posted
on 05 August 2012 - 12:15 PM
pKD46 is a somehow tricky vector ...i regularily exprienced problems with it. It tends to be unstable in certain host backgrounds ...maybe due to basal expression of the araB promoter.
An alternative is to use the pSIM plasmids from the Court lab.
Regards,
p
An alternative is to use the pSIM plasmids from the Court lab.
Regards,
p
#138908 How delete survival gene in bacteria still keeping cell alive.
Posted
on 03 August 2012 - 01:01 PM
prabhubct, on 03 August 2012 - 07:28 AM, said:
How can we select bacterial mutant on growth medium if survival gene is deleted. merely no colony to judge as gene is essential for growth may not be effective. Reason for no colony might be errors in experiment or homologous recombination problem.
what if survival product is not food but some internal machinery of bacteria say DNA polymerase which could not be supplied from outside.
what if survival product is not food but some internal machinery of bacteria say DNA polymerase which could not be supplied from outside.
The problem with deleting or mutating an essential gene does indeed imply that the bacterium wont grow...
See the attachement on how researchers look for essential genes.
And check this: http://www.nature.co...p00125.html#/f2, check figure 1!
Also: to make sure its not because of the medium, you use the same medium as when the gene was not distrupted.
And about the internal machinery of bacteria not be supplied from outside: not sure what you mean by this... If it can not be supplied from outside when you altered the gene.. it will also not be supplied from outside without altering the gene (unless its a gene for a transporter that brings in , for example, a certain nutrient... but then again: it means that the gene you changed is essential for survival).
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#131533 Blunt-ligating dephosphorylated insert into phosphorylated vector
Posted
on 22 March 2012 - 11:44 AM
You can phosphorylate the insert using T4 polynucleotide kinase - note that this is different to the T4 ligase!
#131442 Blunt-ligating dephosphorylated insert into phosphorylated vector
Posted
on 21 March 2012 - 12:00 PM
Your problem probably lies in that you should de-phosphorylate your plasmid so as to prevent self-ligation, seeing as you only cut with one enzyme which can result in re-ligation.
The optimal ligation ratio is usually reaction dependent. You should set up reactions at 3:1, 6:1 and 10:1 to test this. Note that these are molar ratios not ng amounts!
To calculate the molar ratio use this formula:
ng insert = (ratio) x(length of insert in bp/length of plasmid in bp) x ng plasmid
e.g. if you wanted a 20 ng reaction at a 3:1 ratio with an insert of 300 bp and a plasmid of 3000 you would use ng insert = 3 x(300/3000) x 20...
The optimal ligation ratio is usually reaction dependent. You should set up reactions at 3:1, 6:1 and 10:1 to test this. Note that these are molar ratios not ng amounts!
To calculate the molar ratio use this formula:
ng insert = (ratio) x(length of insert in bp/length of plasmid in bp) x ng plasmid
e.g. if you wanted a 20 ng reaction at a 3:1 ratio with an insert of 300 bp and a plasmid of 3000 you would use ng insert = 3 x(300/3000) x 20...
#17740 Equation for calculating insert:vector ratio in ligation reaction
Posted
on 04 March 2009 - 01:16 PM
insert mass in ng = 6 X (insert lenght in bp/vector lenght in bp) X vector mass in ng
You may change the 6 above depending on the actual ratio you want to get (sometimes a 3:1 works best than 6:1)
You may change the 6 above depending on the actual ratio you want to get (sometimes a 3:1 works best than 6:1)
#13566 Handbook of Biological Statistics
Posted
on 28 January 2009 - 12:02 AM
#138834 Alkaline phosphate treatment of vector SalI to ligate it to insert SalI site
Posted
on 02 August 2012 - 10:58 AM
possibly your insert is not cut properly (e.g. SalI has problems cutting PCR products).
I would check if self ligation of your insert is possible by loading a ligation reaction that only contains the insert + ligase on a gel ...should give you multimers of the size of the insert (like a ladder).
Regards,
p
I would check if self ligation of your insert is possible by loading a ligation reaction that only contains the insert + ligase on a gel ...should give you multimers of the size of the insert (like a ladder).
Regards,
p
