BIOL 330                                Lab Final Assessment                          Name:

12/10/2008                                                                                          Lab Section:  WE   TH

 

Show work for all calculation problems.  Answers without some indication of how you arrived at that answer will be given no credit.  Partial credit depends on showing work in a neat and organized way.  Don’t forget the units with answers.  Don’t forget the water in making solutions.  Don’t forget to give the correct significant figures for pipetting volumes.

 

1. Convert the following (show work for partial credit):

 

            23mg/ml = ________________ ng/µl

 

            45µM   =  _________________ mM

 

2.  Tell me what the following settings indicate on each Pipetman Pipet:

 

            P100                                        P1000                                                  P10

               0                                              0                                                          0      

               8                                              3                                                          0

            2….1                                       9….8                                                   6….5

              ^                                                  ^                                                        ^

 

3. Show how to make 75 ml of a 3.5 % agarose solution to make a gel.

 

4. How much of 6mg/ml Proteinase K do you add to 150µl of a DNA solution to give a final

            concentration of 175µg/ml?

 

5. From the stock solutions of 4M NaCl, 20X SSC, 10mg/ml Proteinase K, and 20% SDS, show how to make 300mls of 50mM NaCl, 4X SSC, 200µg/ml Proteinase K, and 1% SDS.

 

6. How much Tris (m.w. = 121g) do you need for 200ml of 10mM solution?

 

7. Set up a restriction digest using Hha I (at 8 U/µl) to digest 6.7µg DNA (480µg/ml) in a

            total volume of 60µl.  Use the maximum number of significant figures for a pipette.

 

8. If a 20-fold dilution of a DNA solution shows an A260 of 0.754 and an A280 of 0.457, what can

            you tell me about the original DNA?

 

9. What is the exact formula for TE?

 

10. What are the sizes (in base pairs, not Kb) of the lambda-Hind III fragments (from smallest to largest)?

 

_____       _____         _____              _____              _____              _____              _____

 

11. Is this wash high or low stringency?  The probe is 350 bases long and 44% (A + T).   

(10X SSC= 2M NaCl + 0.3M Na3Citrate)

 

Tm = 81.5oC + 16.6(log M) + 0.41(%GC) – 0.62 (%formamide) - 500/L

 

Wash Conditions:        Temperature is 65˚C, with 1.5X SSC and 35% formamide

 

12. Each PCR reaction of 50µl will require 40µl of Master Mix and final concentrations of 1X buffer (from 10X), 240µM dNTPs (from 5mM), 3mM MgCl2 (from 25mM), and 1.7 Units of Taq Polymerase (5 U/µl).  Make up 200µl of the MM to deliver these reagents properly.

 

13. Set up a pipetting chart for a PCR experiment with 5 tubes where you will vary the Taq DNA polymerase between 0.5 and 2.5 Units per reaction (in steps of 0.5Units).  Each 50µl PCR reaction tube will also need 15µl of master mix, 2ng of DNA, and two primers (at 0.15µM).  Starting solutions are 5U/µl Taq polymerase, 350µg/ml DNA, and 5µM for each primer.

 

14. How does each of the following help protect DNA during isolation?

 

            A. 55°C

 

            B. EDTA

 

            C. Phenol

 

15. How much 6X loading dye do you add to 20µl of sample to load into a gel?

 

16. Define STAR activity in a restriction enzyme and give two reaction conditions might cause it.

 

17. During the precipitation of DNA, what is the purpose of adding 1/10 volume of sodium

            acetate before adding the ethanol?

 

18. What are two properties of pGEM that helped us during cloning?

 

From the following answer the statements.  Choose best single answer.

            A. luciferase    B. DNase         C. lysozyme     D. proteinase K                        E. ligase                       F. peroxidase       G. Taq polymerase    H. Sal I      I. β galactosidase

 

_____ 21. Coded for in the lux operon

 

_____ 22. Tris at pH 8 helped to stop this from working

 

_____ 23.  Turns XGAL into a blue color

 

_____ 24. Covalently attaches “sticky ends”

 

25. If I added 0.8ml of lysozyme (10mg/ml) to 12mls of bacteria, what is the final concentration

of lysozyme?

 

_____ 26. How did we make DNA on the membrane single-stranded during Southern Blotting?

            A. Used NaCl to transfer DNA                        B. Soaked membrane in SSC

            C. Used H2O2                                       D. None

 

27. What was the “probe” for our PCR-Southern Blotting experiment?  How did we attach an

            enzyme to the probe to find where it was bound on the blot?

 

28. Draw the following reactions as you would expect to see them on a gel.  pGEM is 3200 bases.

            (Hint:  Do lane 3 first)

                                                                                       1       2       3       4       5       6

Lane 1             V. fischeri DNA, genomic

 

Lane 2             V.fischeri DNA cut with Sal I

 

Lane 3             λ DNA, Hind III

 

Lane 4             pGEM, cut with Sal I

 

Lane 5             pGEM

 

Lane 6             PCR product about 700b size

 

29. What happens during each step in a single PCR cycle?  Give step name and temperature as well.

 

30. What is an amplicon (or PCR product)?  Describe what it looks like or its nature.