Knight: Addition of 3' A-overhangs: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
|||
(2 intermediate revisions by 2 users not shown) | |||
Line 13: | Line 13: | ||
==Notes== | ==Notes== | ||
*I have not yet tried this protocol! | *I have not yet tried this protocol! | ||
* An alternative is TdT with ddATP. | |||
==References== | ==References== | ||
#[http://www.invitrogen.com/content/sfs/manuals/topotaseq_man.pdf Invitrogen TOPO TA Cloning® Kit for Sequencing manual] (page 21) | #[http://www.invitrogen.com/content/sfs/manuals/topotaseq_man.pdf Invitrogen TOPO TA Cloning® Kit for Sequencing manual] (page 21) |
Latest revision as of 20:22, 18 October 2008
Overview
This protocol adds 3' A-overhangs to a PCR product rendering it suitable for TA cloning.
Materials
- Taq polymerase
- A heat block equilibrated to 72°C
Procedure
- After amplification with the proofreading polymerase, place vials on ice and add 0.7-1 unit of Taq polymerase per tube. Mix well. It is not necessary to change the buffer. A sufficient number of PCR products will retain the 3´ A-overhangs.
- Incubate at 72°C for 8-10 minutes (do not cycle).
- Place on ice and use immediately in the TOPO cloning reaction.
Notes
- I have not yet tried this protocol!
- An alternative is TdT with ddATP.