Class Notes (808,849)
Canada (493,411)
Biochemistry (847)


2 Pages
Unlock Document

Western University
Biochemistry 2280A
Derek Mc Lachlin

Brandl Lecture 8 Notes 11/26/2012  Some restriction enzymes cut identical sequences, but leave different types of ends o Example: BamHI and NlaIV cut identical sequences, but BamHI cuts to give a 5’ overhangs while NlaIV cuts in the middle to give a blunt end  Some restriction enzymes cut different recognition sequences but leave identical overhanging ends o Example: BamHI and BglII  Q: What is the probability that any random 4 base pair sequence is a HaelIII site (GGCC)? A: 1 in 256 The probability that the 1 position is a G is 1 in 4, the probability that the 2 position is a G is 1 in 4, the rd th probability that the 3 position is a C is 1 in 4, and the probability that the 4 position is C is 1 in 4 ¼ x ¼ x ¼ x ¼ = 1/256  Q: Given that the probability of any 6 base pair sequence being an HincII site is about 1/1000, how many HincII sites are found within the human genome (3 billion base pairs)? A: 3 million (3 x 10^9 / 10^3)  Restriction enzymes are enzymes o Like any other enzymes, each restriction enzyme has preferred conditions in which it functions o These include: temperature, pH, salt concentration  Specific conditions at which they cut DNA  Same properties as enzymes  Restriction enzymes have reaction kinetics o Takes time to cut DNA  We can cut DNA with restriction enzymes – how can we stitch DNA back together? DNA Ligase  DNA ligase = glue for DNA  Will reseal compatible sticky ends and much less efficiently blunt ends  Requires an energy source (ATP)  DNA ligase will find annealed fragments and use energy to form covalent bonds (phosphodiester bonds) to seal up ends that are compatible o Ends must be compatible sticky ends to be ligated  5’ ends on the DNA must have phosphates o Ligation will not work otherwise Steps in Cloning  Digest about 100 ng of YFG and a plasmid vector with the same restriction enzyme or one that gives compatible overhanging sequences  How do we get the restriction enzyme? o Putting EcoRI sites on the cDNa by PCR  Purify your DNA o Often by electrophoresis  Incubate vector (plasmid) and insert (YFG) together in the presence of DNA ligase and ATP o The sticky ends anneal (hybridize) o Ligase forms covalent bonds (phosphodiester bonds) sealing the ends  Now must get the recombinant plasmid into the E. Coli – to do this, we use a process called transformation Transformation  The process by which cells take up DNA from their environment  Natural property of some bacteria o E. coli have to be treated with chemicals to do it well  Mix E. coli with ligated DNA – some of the E. coli take up the plasmids o Cells that have taken up the plasmid
More Less

Related notes for Biochemistry 2280A

Log In


Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.