(PCR)
Line 5: Line 5:
 
* When adding restriction sites to your primers, check that there is sufficient space if that site is near the end of the primer for the enzyme to be able to sufficiently bind and cut. As a general rule, 4-6 bp is a good length to protect your site with although this can vary to be as few as 1bp and as many as 9bp. For more information, visit the NEB information site.
 
* When adding restriction sites to your primers, check that there is sufficient space if that site is near the end of the primer for the enzyme to be able to sufficiently bind and cut. As a general rule, 4-6 bp is a good length to protect your site with although this can vary to be as few as 1bp and as many as 9bp. For more information, visit the NEB information site.
 
* The fact that '''primers are synthesized 3' to 5'''' means that all or nearly all primers are correct and identical at the 3' end, which is important for locating the site of biological synthesis. Synthesized primers typically have a significant error rate, with most 100 bp primers being incorrect in one or more locations. The most common error is a truncation of the 5' end, followed by point deletions of single bases. Long strings of G's are problematic for synthesis.
 
* The fact that '''primers are synthesized 3' to 5'''' means that all or nearly all primers are correct and identical at the 3' end, which is important for locating the site of biological synthesis. Synthesized primers typically have a significant error rate, with most 100 bp primers being incorrect in one or more locations. The most common error is a truncation of the 5' end, followed by point deletions of single bases. Long strings of G's are problematic for synthesis.
* Select a proper polymerase for your application:
+
* Always choose a proper polymerase for your application:
 
[[File:Polymerase_fidelity.jpg|600px|center|Polymerase fidelity comparison]]
 
[[File:Polymerase_fidelity.jpg|600px|center|Polymerase fidelity comparison]]

Revision as of 14:17, 9 April 2015

Experiences

PCR

  • With your low GC, you should also consider lowering your extension (not the annealing) temperature and doubling the extension time. This will allow you to PCR very low GC regions, which otherwise cannot be extended.
  • When adding restriction sites to your primers, check that there is sufficient space if that site is near the end of the primer for the enzyme to be able to sufficiently bind and cut. As a general rule, 4-6 bp is a good length to protect your site with although this can vary to be as few as 1bp and as many as 9bp. For more information, visit the NEB information site.
  • The fact that primers are synthesized 3' to 5' means that all or nearly all primers are correct and identical at the 3' end, which is important for locating the site of biological synthesis. Synthesized primers typically have a significant error rate, with most 100 bp primers being incorrect in one or more locations. The most common error is a truncation of the 5' end, followed by point deletions of single bases. Long strings of G's are problematic for synthesis.
  • Always choose a proper polymerase for your application:
Polymerase fidelity comparison