3' end labelling with tritiated thymidine...

[Rory O'Brien]

Hi Group,

 

I'm looking to try end-tailing an oligonucleotide with terminal transferase
and labeling it with tritium in the process; the tritiated thymidine
products available from Perkin Elmer come as -

 

NET221H - Deoxythymidine 5`-Triphosphate, [Methyl-3H]-, Tetrasodium Salt,
Specific Activity: 10-25Ci (370-925GBq)/mMole, >97%, 1mCi/mL. Packed in
ethanol :water (1:1)

 

NET221X -  Deoxythymidine 5`-Triphosphate, [Methyl-3H]-, Tetrasodium Salt,
Specific Activity: 70-90Ci (2.59-3.33TBq)/mMole, 1mCi/mL.  Packed in ethanol
:water (1:1)

 

NET221A - Deoxythymidine 5`-Triphosphate, [Methyl-3H]-, Tetrasodium Salt,
Specific Activity: 70-90Ci (2.59-3.33TBq)/mMole, 2.5mCi/mL. Packed in 10mM
tricine buffer (pH 7.6)

 

 

Could anyone maybe suggest which thymidine product might be most appropriate
to this application? Detection to be via scintillation counter; one for the
old-school molecular biologists maybe ;-)

 

Cheers,

 

- Rory

 

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[Duncan Clark]

Historians believe that in newspost
<mailman.70.1255061417.1133.methods@...> on Fri, 9 Oct 2009,
Rory O'Brien <rory.obrien@...> penned the following
literary masterpiece:
>Could anyone maybe suggest which thymidine product might be most appropriate
>to this application? Detection to be via scintillation counter; one for the
>old-school molecular biologists maybe ;-)

Highest specific activity would be best so either of the last two.
Whether they are the least expensive is another matter. If you are
simply using it to check the incorporation even the first one should do.
You will just see lower counts.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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[Rory O'Brien-2]

Cheers Duncan,

The question came from a student here who wants to measure uptake of an
oligo into bacterial cells and the effect of a drug on same. The thought was
to label the oligo and measure via scintillation counter. I had thought we
could end tail the oligo with tritiated thymidine but she's now wary of
sticking a whole bunch of nucleotides onto the end of the oligo and the
effect that might have on her uptake experiment. I doubt we'd get a strong
enough signal with just end labeling and I'd rather avoid P32 as we're not
really set up for it here.

Could we do something non-isotopic with digoxygenin do you think? Maybe a
dot blot of lysed bugs and semi-quantitative chemiluminescent detection?

- Rory



Historians believe that in newspost
<mailman.70.1255061417.1133.methods@...> on Fri, 9 Oct 2009,
Rory O'Brien <rory.obrien@...> penned the following
literary masterpiece:
>Could anyone maybe suggest which thymidine product might be most
appropriate
>to this application? Detection to be via scintillation counter; one for the
>old-school molecular biologists maybe ;-)

Highest specific activity would be best so either of the last two.
Whether they are the least expensive is another matter. If you are
simply using it to check the incorporation even the first one should do.
You will just see lower counts.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
_______________________________________________
Methods mailing list
Methods@...
http://www.bio.net/biomail/listinfo/methods


_______________________________________________
Methods mailing list
Methods@...
http://www.bio.net/biomail/listinfo/methods

[Duncan Clark]

Historians believe that in newspost
<mailman.90.1255380772.1133.methods@...> on Tue, 13 Oct 2009,
Rory O'Brien <rory.obrien@...> penned the following literary
masterpiece:
>Could we do something non-isotopic with digoxygenin do you think?

Problem then is, will an oligo with such a big group stuck on the end go
into the cell and if so will such a big group alter the experimental
outcome.

Why not kinase an oligo with gamma labelled 32P ATP?

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
_______________________________________________
Methods mailing list
Methods@...
http://www.bio.net/biomail/listinfo/methods