1. What would happen if you attempted to run DNA on an agarose gel with water instead of TAE or TBE? Why would this be the case?
2. Answer the following questions:
a)Using a DNA sample that is 0.2g/ul, set up a 20-l digestion with Bam HI containing 1 ug DNA, 10 units of enzyme (10,000U/mL) and the appropriate buffer.
b)After digestion you want to stop the reaction by heat inactivating the enzyme. Is this possible and if so at what temperature would you do this?
c)You also want to consider doing a double digestion with 10 units of BamHI and 10 units of BciV1 (5,000U/mL). Set up a 20digestion with Bam HI and BciV1containing 2 ug DNA
3.Design a PCR program for amplification of a 1500-bp fragment from a genomic DNA template. The forward and reverse primers have calculated melting points of 57C and 58C, respectively. Name each step, including any initial or final steps additional to the 3 basic cycles. Do not give any procedures beyond listing the PCR program.
4.While doing a transformation of E. coli with a plasmid your colleague learned that she was urgently needed at home. She asked you to finish the process. She had just heat-shocked the cells at 42°C and placed them on ice. Tell me the steps you would take after this point to complete the transformation so that your friend will be able to check on the results the next day.
5.Increasing the GC content of an oligonucleotide has the effect of
a.increasing melting temperature
b.lowering melting temperature
c.increasing specificity for annealing to the template
d.decreasing specificity for annealing to the template
e.both a and c
6.You performed restriction digestion of the plasmid vector with a unique restriction enzyme and ran a gel for the uncut plasmid mini prep along with linearized plasmid vector. In the picture below, indicate linear, supercoiled and nicked circular version of your plasmid that is ~4kb in size.
Please write it in the provided white space below the band and provide explanation.
7.You carried out gel purification of the plasmid and then you restriction digested with EcoRI. Why was this necessary (what does it accomplish)?
8. What is the major purpose of the first step in the gel purification process?
9. What are the advantages of In-Fusion cloning over traditional cloning? Write the outline of steps (no need to give volume details) you performed for In-Fusion cloning.
10. In a mini prep (plasmid isolation), after the cells have been resuspended, what do the next two steps achieve and how?
11. In general it is best not to add a volume of restriction enzyme to a digestion that represents 25% or more of the total volume. Why not?
12. Unless we can dilute the template DNA for a PCR reaction why would it not be stored in TE (Tris EDTA) buffer. [Why would we not want to add DNA in TE to a PCR reaction
13. Name the essential cofactor (normally present in the ligation buffer) required for T4 DNA ligase activity. How does it help with the ligase function?
14. Design a forward primer and reverse primer to amplify the following p53 gene sequence.
15. Write the steps (and purpose) done for Western blotting.
16. What was the purpose of performing the BCIP test on the E. coli cells grown on High Phosphate and No Phosphate media?
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