Lidstrom:Ligation

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(New page: Back to Protocols From [http://2011.igem.org/Team:Washington/Protocols/Digestion iGem UW 2011]: 7uL insert 1uL vector 1uL T4 ligase buffer 1uL T4 ligase Incubate at...)
Current revision (15:03, 6 March 2012) (view source)
 
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From [http://2011.igem.org/Team:Washington/Protocols/Digestion iGem UW 2011]:
From [http://2011.igem.org/Team:Washington/Protocols/Digestion iGem UW 2011]:
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7uL insert
+
*7uL insert
-
1uL vector
+
*1uL vector
-
1uL T4 ligase buffer
+
*1uL T4 ligase buffer
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1uL T4 ligase
+
*1uL T4 ligase
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Incubate at 16C overnight, or room temperature for 30 minutes to 1 hour.
+
*Incubate at 16oC overnight, or room temperature for 30 minutes to 1 hour.
==To vary vector:insert ratio?==
==To vary vector:insert ratio?==

Current revision

Back to Protocols

From iGem UW 2011:

  • 7uL insert
  • 1uL vector
  • 1uL T4 ligase buffer
  • 1uL T4 ligase
  • Incubate at 16oC overnight, or room temperature for 30 minutes to 1 hour.

To vary vector:insert ratio?

  • You can do this, and thorough people do. Try doing it fast and simple (as iGem does above) before trying laborious/precise calculations & ratios.

To use controls?

  • You can do this, and thorough people do. The most common controls to use are VCL (vector, cut + ligase) and VCNL (vector, cut, no ligase used). These help you assess the frequency of the plasmid backbone's recircularization in the presence and absence of ligase. If you see a lot of colonies in the VCL plate, you should screen more of your colonies because more of them will be false positives.
  • It often doesn't take longer to include these controls, so if you have enough DNA you can do it. But it does require additional antibiotic plates which require labor to make!
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