Richard Lab:Ligation

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(Notes)
(Procedure)
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1.Mix together the following:
1.Mix together the following:
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::a. 5µl digested front part (EcoRI and SpeI).
+
::a. 11µl of deionized water
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::b. 5µl digested rear part (XbaI and PstI).
+
::b. 2µl of Reaction Buffer for T4 Ligase.
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::c. 4µl deionized water.
+
::c. 3µl digested "Vector" (Phosphatased).  
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::d. 3µl digested vector (EcoRI and PstI).  
+
::d. 3µl digested "Insert" (PCR product with DpnI).
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::e. 2µl of Reaction Buffer for T4 Ligase.
+
::e. 1µl of [[T4_DNA_ligase|DNA Ligase]].
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::f. 1µl of [[T4_DNA_ligase|DNA Ligase]].
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2.  Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)<br>
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2.  Incubate at room-temp for 10 mins (or at 4°C overnight)<br>
+
3.  Kill Reaction for 15 mins at 65°C
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3.  Kill Reaction for 15 mins at 80°C
+
===Notes===
===Notes===
* [[User:Michael A. Speer|Mike]] has had problems with blunt end ligation if this reaction is let go for too long.
* [[User:Michael A. Speer|Mike]] has had problems with blunt end ligation if this reaction is let go for too long.
* If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.
* If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.

Revision as of 18:46, 12 January 2011

This protocol is for the typical ligation of DNA done in the Richard Lab.

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. 3µl digested "Vector" (Phosphatased).
d. 3µl digested "Insert" (PCR product 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

Notes

  • Mike has had problems with blunt end ligation if this reaction is let go for too long.
  • 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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