ta-cloning vector

dreamer at super.zippo.com dreamer at super.zippo.com
Tue Oct 1 10:36:37 EST 1996


Lynne_M_Roxbee_Cox <Lynne_M_Roxbee_Cox at sbphrd.com> wrote:


>I don't think this article was in the Euro edition (Jan/Feb 1996) of 
>Biotechniques.

>Could you let us know what it said please.

>Thank you,

>Lynne
>-- 

- Cut vector (10ug)  with EcoRV and treat for 2h in PCR buffer (100ul)
with dTTP (2mM) and 5u of Taq pol, at 72C (authors use Life
Technologies buffer and Taq)
- Perform phenol/chloroform extraction and ethanol precipitation. Dry
the pellet and resusupend in small volume of water
- Ligate in a volume of 50ul for 16h using 2U ligase (authors use Life
Technologies ligase buffer)
- Run agarose gel and cut the sharp band of unligated linearized
vector (most of it contains T-overhangs) away from ligated vector
molecules (which ligated because they didn't have the T-overhang)
- Purify DNA from the gel slice (authors use GeneClean)
- Run a sample on a gel to quantify.

Authors get cloning efficiency of PCR products up to 80%







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