7,287
edits
Knight:Annealing and primer extension with Taq polymerase: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Knight:Annealing and primer extension with Taq polymerase (view source)
Revision as of 16:49, 19 October 2006
, 19 October 2006→Notes
No edit summary |
(→Notes) |
||
| Line 31: | Line 31: | ||
*For oligos greater than 50-60 bp in length, there can often be problems with errors or deletions in the primers. Therefore, it might be worth ordering your primers with an extra purification step such as PAGE. [https://catalog.invitrogen.com/index.cfm?fuseaction=customerSite.primersHome Invitrogen custom primers] offers this service for an extra fee. | *For oligos greater than 50-60 bp in length, there can often be problems with errors or deletions in the primers. Therefore, it might be worth ordering your primers with an extra purification step such as PAGE. [https://catalog.invitrogen.com/index.cfm?fuseaction=customerSite.primersHome Invitrogen custom primers] offers this service for an extra fee. | ||
*'''[[User:Rshetty|RS]] 21:30, 14 June 2006 (EDT)''': I recently have been trying to make a promoter via this method. I sequenced 3 different clones and all of them had a 1bp deletion in the same position. This was despite the fact that I had ordered my primers to be PAGE purified. I emailed Invitrogen and they said that they will resynthesize and ship me a new primer free of charge. We'll see what happens. | *'''[[User:Rshetty|RS]] 21:30, 14 June 2006 (EDT)''': I recently have been trying to make a promoter via this method. I sequenced 3 different clones and all of them had a 1bp deletion in the same position. This was despite the fact that I had ordered my primers to be PAGE purified. I emailed Invitrogen and they said that they will resynthesize and ship me a new primer free of charge. We'll see what happens. | ||
*'''[[User:Rshetty|Reshma]] 19:49, 19 October 2006 (EDT)''': Tom suggests considering how much enzyme you use relative to how much primer. Supposedly, polymerase does not necessarily fall off the ends of the DNA which means in 2-3 cycles, very few of your annealed primers will become completely double-stranded (as this requires two polymerases to bind to the same annealed primer molecule and extend). This lack of complete double stranding could increase the potential for errors. | |||
==References== | ==References== | ||
<biblio> | <biblio> | ||
#Stemmer-Gene-1995 pmid=7590320 | #Stemmer-Gene-1995 pmid=7590320 | ||
</biblio> | </biblio> | ||
