Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces...
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Transcript of Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces...
![Page 1: Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces of DNA found in bacteria What are restriction enzymes?](https://reader036.fdocuments.in/reader036/viewer/2022062423/56649f4f5503460f94c71ac9/html5/thumbnails/1.jpg)
Restriction Analysis of PlasmidsRestriction Analysis of Plasmids
Lab Details
![Page 2: Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces of DNA found in bacteria What are restriction enzymes?](https://reader036.fdocuments.in/reader036/viewer/2022062423/56649f4f5503460f94c71ac9/html5/thumbnails/2.jpg)
Memory Jog
• What are plasmids?– Small, circular pieces of DNA found in bacteria
• What are restriction enzymes?– Enzymes that cut DNA at specific sequences
• How can you create a recombinant plasmid?– Cut gene of interest and plasmid with the same
restriction enzyme– Mix together and seal with ligase
![Page 3: Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces of DNA found in bacteria What are restriction enzymes?](https://reader036.fdocuments.in/reader036/viewer/2022062423/56649f4f5503460f94c71ac9/html5/thumbnails/3.jpg)
Plasmid Mapping
• In order to use a plasmid to create recombinant plasmids, we need to find out – Which restriction enzymes will cut a particular
plasmid– How many places a restriction enzyme will cut
a particular plasmid– How far apart restriction sites are
![Page 4: Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces of DNA found in bacteria What are restriction enzymes?](https://reader036.fdocuments.in/reader036/viewer/2022062423/56649f4f5503460f94c71ac9/html5/thumbnails/4.jpg)
Sample Problem #1
• Suppose 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.
BamHI
+ BamHI
Plasmid A
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Sample Problem #2• Suppose you have another plasmid called
plasmid B that has 2 restriction sites for BamHI:
BamHI (0 bp)
BamHI (700 bp)
One restriction site is arbitrarily determined to be the 0 point
The other restriction sites are given in reference to the 0 point
Plasmid B1250 bp
![Page 6: Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces of DNA found in bacteria What are restriction enzymes?](https://reader036.fdocuments.in/reader036/viewer/2022062423/56649f4f5503460f94c71ac9/html5/thumbnails/6.jpg)
• How many fragments will result from digesting this plasmid with BamHI?– 2 different fragments
• How big are the fragments?– 700 bp and 550 bp
BamHI (0 bp)
BamHI (700 bp) 700 bp
550 bp
![Page 7: Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces of DNA found in bacteria What are restriction enzymes?](https://reader036.fdocuments.in/reader036/viewer/2022062423/56649f4f5503460f94c71ac9/html5/thumbnails/7.jpg)
Lab
• Your task: Analyze 2 different plasmids (B, S or pGFP) to determine– The total size (in bp) of each plasmid– How many restriction sites each plasmid has for
the enzyme HindIII or another restriction enzyme
– How far apart the restriction sites are from one another (if there is more than one)
![Page 8: Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces of DNA found in bacteria What are restriction enzymes?](https://reader036.fdocuments.in/reader036/viewer/2022062423/56649f4f5503460f94c71ac9/html5/thumbnails/8.jpg)
HindIII
• HindIII recognizes the following sequence:
![Page 9: Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces of DNA found in bacteria What are restriction enzymes?](https://reader036.fdocuments.in/reader036/viewer/2022062423/56649f4f5503460f94c71ac9/html5/thumbnails/9.jpg)
General Steps
• Monday: Digest each of the DNA samples with enzyme for 30 minutes
• Tues/Wed: Run the DNA samples on a gel
• Thurs: Analyze gels
![Page 10: Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces of DNA found in bacteria What are restriction enzymes?](https://reader036.fdocuments.in/reader036/viewer/2022062423/56649f4f5503460f94c71ac9/html5/thumbnails/10.jpg)
Uncut plasmids
• Uncut plasmids will also be run as a control (labeled __“0”)– no HindIII added
• An uncut plasmid is still circular won’t move through the gel in the same way a linear molecule would
• Running these controls helps us tell which bands in gel are due to uncut plasmids (so we don’t count them as fragments).
![Page 11: Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces of DNA found in bacteria What are restriction enzymes?](https://reader036.fdocuments.in/reader036/viewer/2022062423/56649f4f5503460f94c71ac9/html5/thumbnails/11.jpg)
Uncut Plasmid Forms
• Uncut plasmid are most likely to exist in one of 3 forms:– Supercoiled– Nicked– Multimer
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Supercoiled Plasmid
• When the plasmid gets twisted up
• Moves faster through the gel than other forms of uncut plasmid because they are so compact
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Nicked circles
• Sometimes the plasmids get partly “nicked” (cut) by handling and refreezing
• Causes it to unwind can’t move through the gel as easily as the supercoiled form
![Page 14: Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces of DNA found in bacteria What are restriction enzymes?](https://reader036.fdocuments.in/reader036/viewer/2022062423/56649f4f5503460f94c71ac9/html5/thumbnails/14.jpg)
Multimers
• Sometimes plasmids get linked together when they are being copied in a bacteria
• This multimer has twice as much DNA so it moves slower through the gel.
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Uncut Plasmids on a Gel
• When you run the uncut plasmids on a gel, you will get a pattern of bands (at least 3-4)
• Which band of DNA is – the supercoiled plasmids?– Nicked circles?– Multimers?
supercoiled
-
+
Nicked circle
Multimer
![Page 16: Restriction Analysis of Plasmids Lab Details Memory Jog What are plasmids? –Small, circular pieces of DNA found in bacteria What are restriction enzymes?](https://reader036.fdocuments.in/reader036/viewer/2022062423/56649f4f5503460f94c71ac9/html5/thumbnails/16.jpg)
• Now, walk through Monday’s Procedure
• Homework = Set up Lab Notebook– Write a specific title (hint: plasmid and Enzyme
names…)– Write an Introduction– Paste in Procedure (both pages)– make a flowchart of procedure