Corum:Gel Purification: Difference between revisions

Revision as of 15:14, 28 June 2012

Vector construction by ligation of dsDNA linker into a dsDNA vector backbone with compatible restriction sites.

ssDNA linker oligonucleotides OR ssDNA primer oligonucleotides and linker DNA template
PCR reaction components
Gel electrophoresis components
Gel extraction kit
PCR purification kit
Ligation reaction components
Transformation components
Miniprep kit

Design linker and vector backbone with compatible overhanging restriction sites.
Construct linker by
- Hybridization of ssDNA oligonucleotides (<100bp)
- OR PCR amplification of linker DNA template with ssDNA oligonucleotide primers (>100bp) followed directly by double digestion with restriction enzymes (heat inactivate 80 °C 20 min) and PCR purification of the digestion product. Elute in 100 μL sterile H₂O.
In parallel, construct vector backbone by double digestion with restriction enzymes (30 μL reaction volume), followed by gel extraction of the backbone, leaving the cut-out piece behind. Elute in 100 μL sterile H₂O.
Perform ligation on the linker and vector backbone.
Transform 3 μL ligation product into competent cells.
Grow 4-6 parallel overnight minicultures from competent cells.
Perform miniprep plasmid isolation on the minicultures.
Sequence the miniprep products.
Verify the constructs by comparing the expected linker sequence with the actual linker sequence. Retain good clones. Discard bad clones.

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@@ Line 16: / Line 16: @@
 ==Procedure==
-#
+# Design linker and vector backbone with compatible overhanging restriction sites.
+# Construct linker by
+#* Hybridization of ssDNA oligonucleotides (<100bp)
+#* OR PCR amplification of linker DNA template with ssDNA oligonucleotide primers (>100bp) followed directly by double digestion with restriction enzymes (heat inactivate 80 °C 20 min) and PCR purification of the digestion product. Elute in 100 μL sterile H<sub>2</sub>O.
+# In parallel, construct vector backbone by double digestion with restriction enzymes (30 μL reaction volume), followed by gel extraction of the backbone, leaving the cut-out piece behind. Elute in 100 μL sterile H<sub>2</sub>O.
+# Perform ligation on the linker and vector backbone.
+# Transform 3 μL ligation product into competent cells.
+# Grow 4-6 parallel overnight minicultures from competent cells.
+# Perform miniprep plasmid isolation on the minicultures.
+# Sequence the miniprep products.
+# Verify the constructs by comparing the expected linker sequence with the actual linker sequence. Retain good clones. Discard bad clones.
 ==Notes==