Richard Lab:Ligation

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(Procedure)
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1.Mix together the following:
1.Mix together the following:
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::a. 9µl of digested gel el extracted insert
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::a. 5µl digested front part (EcoRI and SpeI).
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::b. 5µl of digested gel extracted vector
+
::b. 5µl digested rear part (XbaI and PstI).
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::c. 2µl of Reaction Buffer (or any NEBuffer)
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::c. 4µl deionized water.
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::d. 2µl of  10mM ATP solution (only if NEBuffer was used)
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::d. 3µl digested vector (EcoRI and PstI).
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::e.1µl of [[T4_DNA_ligase|DNA Ligase]]
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::e. 2µl of Reaction Buffer for T4 Ligase.
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::f. 1µl of PEG solution (optional)
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::f. 1µl of [[T4_DNA_ligase|DNA Ligase]].
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::g. Water to bring up to 20µl
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2.  Incubate at room-temp for 10 mins (or at 4°C overnight)
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2.  Incubate at room-temp for 30-120 mins (or at 16°C overnight)
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3.  Kill Reaction for 15 mins at 80°C
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3.  Kill Reaction for 15 mins at 70°C
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===Notes===
===Notes===
* [[User:Michael A. Speer|Mike]] has achieved good results with leaving the ligation in a drawer overnight, but this should not be done if using PEG to speed up the ligation.
* [[User:Michael A. Speer|Mike]] has achieved good results with leaving the ligation in a drawer overnight, but this should not be done if using PEG to speed up the ligation.
* 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 14:59, 6 April 2010

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. 5µl digested front part (EcoRI and SpeI).
b. 5µl digested rear part (XbaI and PstI).
c. 4µl deionized water.
d. 3µl digested vector (EcoRI and PstI).
e. 2µl of Reaction Buffer for T4 Ligase.
f. 1µl of DNA Ligase.

2. Incubate at room-temp for 10 mins (or at 4°C overnight) 3. Kill Reaction for 15 mins at 80°C

Notes

  • Mike has achieved good results with leaving the ligation in a drawer overnight, but this should not be done if using PEG to speed up the ligation.
  • 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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