Cloning CRISPR-Cas Plasmids: Difference between revisions

Line 34:

===Verification===

* Miniprep the transformed clones and digest 1 ug of purified plasmid for 1h with AgeI and BbsI in NEB Buffer 1.1 and then run on an agarose gel. Empty vectors will yield a ~1kb fragment, wheras vectors with insert will only be cut once. This is because the cloning removes the BbsI site.

* Send clones with insert for sequencing with the hU6 sequencing primer. Align those clones with the empty vector full sequence or the hU6 sequencing of the empty vector, available at http://www.addgene.org/42335/sequences/. Doublecheck that your insert is correct, and is oriented in the correct manner.

* Send clones with insert for sequencing with the hU6 sequencing primer. Go to http://bridgeslab.sph.umich.edu/protocols/index.php/Submitting_Plasmids_for_Sequencing for further details. Align those clones with the empty vector full sequence or the hU6 sequencing of the empty vector, available at http://www.addgene.org/42335/sequences/. Doublecheck that your insert is correct, and is oriented in the correct manner.

@@ Line 34: / Line 34: @@
 ===Verification===
 * Miniprep the transformed clones and digest 1 ug of purified plasmid for 1h with AgeI and BbsI in NEB Buffer 1.1 and then run on an agarose gel.  Empty vectors will yield a ~1kb fragment, wheras vectors with insert will only be cut once.  This is because the cloning removes the BbsI site.
-* Send clones with insert for sequencing with the hU6 sequencing primer.  Align those clones with the empty vector full sequence or the hU6 sequencing of the empty vector, available at http://www.addgene.org/42335/sequences/.  Doublecheck that your insert is correct, and is oriented in the correct manner.
+* Send clones with insert for sequencing with the hU6 sequencing primer. Go to http://bridgeslab.sph.umich.edu/protocols/index.php/Submitting_Plasmids_for_Sequencing for further details.  Align those clones with the empty vector full sequence or the hU6 sequencing of the empty vector, available at http://www.addgene.org/42335/sequences/.  Doublecheck that your insert is correct, and is oriented in the correct manner.