Richard Lab:Ligation: Difference between revisions
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Back to [[Richard_Lab:protocols | Protocols]] | Back to [[Richard_Lab:protocols | Protocols]] | ||
<center> | <center> | ||
'''This protocol is for the typical ligation of DNA done | '''This protocol is for the typical ligation of DNA done during [[Richard Lab:Amplified insert assembly|Amplified Insert Assembly]]. | ||
'''The [[DNA_ligation | consensus Ligation protocol]] should be consulted if deviating from the procedure outlined here.'''</center> | '''The [[DNA_ligation | consensus Ligation protocol]] should be consulted if deviating from the procedure outlined here.'''</center> | ||
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::a. 11µl of deionized water | ::a. 11µl of deionized water | ||
::b. 2µl of Reaction Buffer for T4 Ligase. | ::b. 2µl of Reaction Buffer for T4 Ligase. | ||
::c. | ::c. 2µl digested "Vector" (Phosphatased). | ||
::d. | ::d. 4µl digested "Insert" (PCR product digested with DpnI). | ||
::e. 1µl of [[T4_DNA_ligase|DNA Ligase]]. | ::e. 1µl of [[T4_DNA_ligase|DNA Ligase]]. | ||
2. Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)<br> | 2. Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)<br> | ||
Latest revision as of 12:13, 29 March 2011
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This protocol is for the typical ligation of DNA done during Amplified Insert Assembly.
The consensus Ligation protocol should be consulted if deviating from the procedure outlined here.Procedure
1.Mix together the following:
- a. 11µl of deionized water
- b. 2µl of Reaction Buffer for T4 Ligase.
- c. 2µl digested "Vector" (Phosphatased).
- d. 4µl digested "Insert" (PCR product digested with DpnI).
- e. 1µl of DNA Ligase.
2. Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)
3. Kill Reaction for 15 mins at 65°C Do not forget this step
Notes
- If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.
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