Recombinant DNA Techniques chapter 18
Recombinant DNA Techniques chapter 18. Part I. techniques and their applications. Restriction Digest (to be done in lab) Southern Blot Northern Blot Western Blot Cloning. 1. Restriction Digest: The Cutting of DNA. Endonucleases =. Restricition Enzymes. EXAMPLES OF DNA AGAROSE GELS.
Recombinant DNA Techniques chapter 18
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Presentation Transcript
techniques and their applications. • Restriction Digest (to be done in lab) • Southern Blot • Northern Blot • Western Blot • Cloning
1. Restriction Digest: The Cutting of DNA Endonucleases = Restricition Enzymes
EXAMPLES OF DNA AGAROSE GELS Ladder, marker or standard of known DNA sizes
Wild type Hb Sickle cell Hb Restriction enzyme 1 band is too small to view here) 2 3 Restriction enzyme loss of cutting site
Wt= wildtype M= mutated M M Wt Wt Wt M
An Application to Forensic & Paternity Studies 2. The Southern Blot
1. Restriction Digest & gel electrophoresis TRANSFER DNA crime scene DNA suspect 2 DNA suspect 1 DNA victim
You must understand the reason for the “probe”!
So sometimes you have to really search for the differences between DNA.
From: Restriction digest To result SUMMARY OF A SOUTHERN BLOT
Suspect 1 Suspect 2 Crime scene Victim Back to our crime scene
Techniques also are available to measure RNA and Protein 3. Northern Blot for RNA 4. Western Blot for Protein
Western Summary: Blotting techniques with probes Southern Northern
5. Technique: Cloning of specific genes or DNA fragments = restriction enzyme + = ligase
Cut vector Cut foreign DNA or gene Paste together
2 different antibiotic resistant genes Bacteria are now resistant to 2 antibiotics
Cut DNA here & insert foreign DNA
Will your transformed colonies with the insert be white or blue?
