10X ligation buffer ligase 1 vector DNA insert DNA H 2 O. 10 µl Total Volume. 10X ligation buffer ligase 1 vector DNA insert DNA
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1 Biol/Chem 475 S07 Study problems for quiz 1 See also questions posed in lab handouts including ligase handout Answers to questions 1&2 included at the end of this document. 1. You plan to clone a 1.0 kb restriction fragment into a 4.0 kb vector. You want your ligation mixture to have a vector DNA concentration of 2 µg/ml and a 1:2 molar ratio of vector to insert (1 vector : 2 insert ). The stock concentration of fragment DNA is 10 ng/µl and linearized vector DNA is 400 ng/µl. Work up a recipe for this ligation reaction. Show your calculations. 10X ligation buffer vector DNA insert DNA H 2 O 20 µl Total Volume 2. You plan to clone a 200 bp PCR product into a 5.0 kb vector. You want your ligation mixture to have a vector DNA concentration of 2.5 µg/ml and a 1:1 molar ratio of vector to insert (1 vector : 1 insert ). The concentration your PCR product is 0.5 ng/µl and the vector DNA is 500 ng/µl. Work up a recipe for this ligation reaction. Show your calculations. 10X ligation buffer vector DNA insert DNA H 2 O 10 µl Total Volume 1
2 3. Agarose gel stained with ethidium bromide kb a b Lane 1: lambda DNA cut with HindIII (size standards) Lane 2: plasmid DNA 6 kb in size (not digested with a restriction enzyme) a. In one sentence or with a drawing, explain why there are two bands in lane 2. b. Why are there no bands at 6 kb? ( One sentence.) 4. This photograph shows the results of a total nucleic acid analysis of various E. coli cultures generated during the subcloning experiment performed this quarter. By each arrow, indicate the specific identity of the nucleic acid molecule. NOTE: DNA and RNA are not sufficient answers. Account for differences in migration of nucleic acids with the same identity. Lane 1: Hi Lo size standards Lane 2: untransformed cells Lane 3: cells transformed with pgem vector Lane 4-8: cells transformed with ligation mix 2
3 5. One day you and your lab partner set up a restriction digest to cut out the insert fragment (2.2 kb) from a recombinant plasmid.the recombinant plasmid is 6.6 kb in length. You make the following mix: 5 µl plasmid DNA at 1µg/µl in 10 mm Tris & 5mM EDTA 1 µl 10X buffer (10X = Tris, DTT and 40 mm MgCl) 1 µl enzyme at 4000 units/ml 3 µl water 10 You put your restriction digest in the water bath for 2 hours. At the end of this time period, you notice the water bath was at 31 o C. You take an aliquot and run it on a gel kb a c d lane 1:! Hind III lane 2: uncut plasmid 4.4 b e f lane 3: plasmid digest 2.3 g 2.0 5a. Examine lanes 2 and 3 carefully. Explain what each of the bands (a - g) represents. 5b. What went wrong with this experiment? You set up this experiment again and this time you make sure the incubator is at 37 o C. You check the digest and it looks the same as before. What changes in the protocol should you have made? 3
4 6. a. Your lab partner sets up the following subcloning experiment: First he cuts the vector with a restriction enzyme that recognizes the following sequence and cuts where the arrow indicates: 5 G GTACC 3 His insert DNA is rhinoceros genomic DNA cut with an enzyme that has the following specificity: 5 GCATG C 3 He sets up a standard sticky end ligation, etc. He fails to get any recombinants (vector plus rhinoceros DNA) and he comes to you for help. What do you tell him. You must draw a diagram of the problem to get full credit for this question. Be sure to label 5 and 3 ends. b. You suggest that he reprep his genomic DNA and cut with a different enzyme. Which of the following will work? Draw a diagram to explain your choice 1) G GTACC 2) A GTACT 3) AGTAC T GGT ACC 7. Cloning PCR products: One strategy for cloning PCR products takes advantage of a peculiarity of some thermostable polymerases: the addition of a single A (NOT template encoded) to the ends of the DNA strands generated during PCR. These are called A overhangs. a. A standard plasmid cloning vector can be modified to take advantage of this neurotic behavior of the polymerase. Draw a picture of a linearized cloning vector that could be used in a sticky end ligation of a generic PCR product Be sure to show both ends of the vector and the PCR product and how they will come together. Label all 3 and 5 ends. Don t worry about how the linearized cloning vector is generated-- just show me what would work. If your diagram is messy or unreadable, I won t grade it. 4
5 7 b. Compare this type of sticky end ligation with what you did in class. There are a couple significant differences. List two and then indicate whether it is an advantage or a disadvantage over what you did in class and the briefly explain Answers to selected questions Problem 1 10X ligation buffer 2 vector DNA 1* insert DNA 2 H 2 O µl Total Volume * 1/10 dilution of vector DNA [other dilutions also OK] Problem 2 10X ligation buffer 1 vector DNA 1* insert DNA 2 H 2 O 5 10 µl Total Volume * 1/20 dilution of vector DNA [other dilutions also OK] 5
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