2Cut vector and insert with restriction enzymes01:27
3Purify DNA fragments by gel electrophoresis02:36
4Assemble recombinant DNA with ligase03:50
5Transform bacteria with recombinant DNA04:36
6Select transformed cells using antibiotics05:15
7Verify clone integrity by sequencing05:30
Cell BiologyYouTube (Curated Tutorials)
Molecular Cloning explained for Beginners
Protocol
Difficulty
intermediate
Steps
1
Prepare the vector backbone
Identify and prepare the vector DNA (typically a plasmid) containing essential elements like antibiotic resistance genes, origin of replication, promoter regions, and restriction sites where the insert will be cloned.
▶ 00:20
2
Cut vector and insert with restriction enzymes
Use restriction enzymes (molecular scissors) to cleave both the vector backbone and the prepared insert DNA at matching restriction sites, generating compatible sticky ends for assembly.
▶ 01:27
3
Purify DNA fragments by gel electrophoresis
Run the digested DNA samples through an agarose gel matrix under voltage to separate fragments by size, then extract and isolate the desired vector and insert bands from the gel.
▶ 02:36
4
Assemble recombinant DNA with ligase
Incubate the purified vector and insert together with DNA ligase enzyme to join the complementary sticky ends, creating recombinant DNA molecules.
▶ 03:50
5
Transform bacteria with recombinant DNA
Introduce the recombinant DNA into competent bacterial cells (e.g., E. coli) where the plasmids will be taken up and replicated to produce multiple copies of the cloned DNA.
▶ 04:36
6
Select transformed cells using antibiotics
Grow bacteria on antibiotic-containing media to identify and isolate only the cells that have successfully taken up the recombinant DNA, as they carry the antibiotic resistance gene.
▶ 05:15
7
Verify clone integrity by sequencing
Confirm successful cloning by performing PCR and DNA sequencing on the isolated clones to verify that the desired DNA fragment is present and correctly assembled.