2 Memory Jog What are plasmids? What are restriction enzymes? Small, circular pieces of DNA found in bacteriaWhat are restriction enzymes?Enzymes that cut DNA at specific sequencesHow can you create a recombinant plasmid?Cut gene of interest and plasmid with the same restriction enzymeMix together and seal with ligase
3 Plasmid MappingIn order to use a plasmid to create recombinant plasmids, we need to find outWhich restriction enzymes will cut a particular plasmidHow many places a restriction enzyme will cut a particular plasmidHow far apart restriction sites are
4 Sample Problem #1BamHI+ BamHISuppose you have a plasmid called plasmid A with the one restriction site for BamHI.If you cut this plasmid with BamHI, how many fragments of DNA will you get? (think carefully!)One– cutting the plasmid linearizes it.Plasmid A
5 Sample Problem #2Suppose you have another plasmid called plasmid B that has 2 restriction sites for BamHI:BamHI (0 bp)BamHI (700 bp)The other restriction sites are given in reference to the 0 pointOne restriction site is arbitrarily determined to be the 0 pointPlasmid B1250 bp
6 How many fragments will result from digesting this plasmid with BamHI? 2 different fragmentsHow big are the fragments?700 bp and 550 bpBamHI (0 bp)BamHI (700 bp)700 bp550 bp
7 Lab Your task: Analyze 3 different plasmids (M, B, S) to determine The total size (in bp) of each plasmidHow many restriction sites each plasmid has for the enzyme HindIIIHow far apart the restriction sites are from one another (if there is more than one)
8 HindIIIHindIII recognizes the following sequence:
9 General StepsMonday: Digest each of the DNA samples with HindIII for 30 minutesTues/Wed: Run the DNA samples on a gelThurs: Analyze gels
10 Uncut plasmidsUncut plasmids will also be run as a control (M0, B0, S0)– no HindIII addedAn uncut plasmid is still circular won’t move through the gel in the same way a linear molecule wouldRunning these controls allows us to know which bands in gel are due to uncut plasmids.
11 Uncut Plasmid FormsUncut plasmid are most likely to exist in one of 3 forms:SupercoiledNickedMultimer
12 Supercoiled Plasmid When the plasmid gets twisted up Moves faster through the gel than other forms of uncut plasmid because they are so compact
13 Nicked circlesSometimes the plasmids get partly “nicked” (cut) by handling and refreezingCauses it to unwind can’t move through the gel as easily as the supercoiled form
14 MultimersSometimes plasmids get linked together when they are being copied in a bacteriaThis multimer has twice as much DNA so it moves slower through the gel.
15 -+Uncut Plasmids on a GelWhen you run the uncut plasmids on a gel, you will get a pattern of bands (at least 3-4)Which band of DNA isthe supercoiled plasmids?Nicked circles?Multimers?MultimerNicked circlesupercoiled
16 Pre Lab Quiz tomorrow! Now, walk through Monday’s Procedure Homework = Set up Lab NotebookWrite a specific title (hint: plasmid and Enzyme names…)Write an IntroductionPaste in Procedure (both pages), make a flowchart of procedurePre Lab Quiz tomorrow!