10 replies to this topic

[HN1000]

Hi,
Please tell me the role of DITHIOTHREITOL in PCR.

Thanks

[green]

check this paper

Biochem Mol Biol Int. 1998 Jan;44(1):157-63.
Additive effects of bovine serum albumin, dithiothreitol, and glycerol on PCR.

[HN1000]

green, on Jul 10 2004, 09:21 AM, said:

check this paper

Biochem Mol Biol Int.  1998 Jan;44(1):157-63. 
Additive effects of bovine serum albumin, dithiothreitol, and glycerol on PCR.

Thanks Green.
I try to find the article you mentioned.

[HN1000]

green, on Jul 10 2004, 09:21 AM, said:

check this paper

Biochem Mol Biol Int.  1998 Jan;44(1):157-63. 
Additive effects of bovine serum albumin, dithiothreitol, and glycerol on PCR.

Dear green,
I couldn't find the article. would you please tell me another related article or alternatives?

thanks,
HN1000

[HN1000]

Help again!
I want to know or find detais about Dithiothreitol=DTT and its effect on PCR.
Any articles, online or paper?

thanks

[kawaka]

DTT (dithiothreitol) is used to reduce disulfide bonds quantitatively and maintain monothiols in the reduced state. At low concentrations, DTT stabilizes enzymes and other proteins which possess free sulfhydryl groups and has been shown to restore activity lost by oxidation of these groups in vitro.

DTT is mainly used as a stablizer in PCR. As you can find that many taq polymerases are in DTT containing buffer.

[HN1000]

Dear kawaka,
Thank you very much for your useful information.
But I try to find an article which compare PCR with and without DTT. If you know please point me to that article.

HN1000

[rmbio]

kawaka, on Jul 18 2004, 05:23 AM, said:

DTT (dithiothreitol) is used to reduce disulfide bonds quantitatively and maintain monothiols in the reduced state. At low concentrations, DTT stabilizes enzymes and other proteins which possess free sulfhydryl groups and has been shown to restore activity lost by oxidation of these groups in vitro.

DTT is mainly used as a stablizer in PCR. As you can find that many taq polymerases are in DTT containing buffer.

But what about the proteins which contain disulphide linkage/s in their native form? Wouldn't reducing these hamper their enzymatic activity?

[genehunter]

Its concentration dependent.

[AquaPlasmid]

Interesting, a couple weeks ago I ran out of Taq and got a vial of Taq expired in 2005 from next door and it didn't work. I put in some fresh DTT to 1mM and ban it worked nicely.

Enzymes could form incorrect SS bonds during storage resulting in loss of activity. DTT could leak over time from the Taq prep, getting some fresh DTT helps reduce the SS bonds and allows the protein to fold back into its correct conformation (energy minimum) and form the correct the SS bonds and regain its activity.

Take home - don't trash your expired enzymes just yet.

[hanming86]

AquaPlasmid, on Mar 6 2009, 02:56 PM, said:

Interesting, a couple weeks ago I ran out of Taq and got a vial of Taq expired in 2005 from next door and it didn't work. I put in some fresh DTT to 1mM and ban it worked nicely.

Enzymes could form incorrect SS bonds during storage resulting in loss of activity. DTT could leak over time from the Taq prep, getting some fresh DTT helps reduce the SS bonds and allows the protein to fold back into its correct conformation (energy minimum) and form the correct the SS bonds and regain its activity.

Take home - don't trash your expired enzymes just yet.

NIce one Aqua. If properly stored, enzyme really oould last for a long long time.