Richard Lab:Ligation

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(New page: '''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 out...)
(Procedure)
Line 9: Line 9:
::c. 2µl of Reaction Buffer (or any NEBuffer)
::c. 2µl of Reaction Buffer (or any NEBuffer)
::d. 2µl of  10mM ATP solution (only if NEBuffer was used)
::d. 2µl of  10mM ATP solution (only if NEBuffer was used)
-
::e.1µl of DNA Ligase
+
::e.1µl of [[T4_DNA_ligase|DNA Ligase]]
::f. 1µl of PEG solution (optional)
::f. 1µl of PEG solution (optional)
::g. Water to bring up to 20µl
::g. Water to bring up to 20µl
2.  Incubate at room-temp for 30-120 mins (or at 16°C overnight)
2.  Incubate at room-temp for 30-120 mins (or at 16°C overnight)
3.  Kill Reaction for 15 mins at 70°C
3.  Kill Reaction for 15 mins at 70°C
-
 
===Notes===
===Notes===
* [[User:Michael A. Speer|Mike]] has achieved good results with leaving the ligation in a drawer overnight.
* [[User:Michael A. Speer|Mike]] has achieved good results with leaving the ligation in a drawer overnight.

Revision as of 14:55, 18 June 2009

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.</center>

Procedure

1.Mix together the following:

a. 9µl of digested gel el extracted insert
b. 5µl of digested gel extracted vector
c. 2µl of Reaction Buffer (or any NEBuffer)
d. 2µl of 10mM ATP solution (only if NEBuffer was used)
e.1µl of DNA Ligase
f. 1µl of PEG solution (optional)
g. Water to bring up to 20µl

2. Incubate at room-temp for 30-120 mins (or at 16°C overnight) 3. Kill Reaction for 15 mins at 70°C

Notes

  • Mike has achieved good results with leaving the ligation in a drawer overnight.
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